18891, a novel human lipase

ABSTRACT

The present invention relates to a newly identified human lipase belonging to the family of mammalian lipases. The invention also relates to polynucleotides encoding the lipase. The invention further relates to methods using the lipase polypeptides and polynucleotides as a target for diagnosis and treatment in lipase-mediated or -related disorders. The invention further relates to drug-screening methods using the lipase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the lipase polypeptides and polynucleotides. The invention further relates to procedures for producing the lipase polypeptides and polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/434,613, filed Nov. 5, 1999 now U.S. Pat. No. 6,337,187 issued Jan.8, 2002, which is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

The present invention relates to a newly identified human lipase. Theinvention also relates to polynucleotides encoding the human lipase. Theinvention further relates to methods using the lipase polypeptides andpolynucleotides as a target for diagnosis and treatment inlipase-mediated or -related disorders. The invention further relates todrug-screening methods using the lipase polypeptides and polynucleotidesto identify agonists and antagonists for diagnosis and treatment. Theinvention further encompasses agonists and antagonists based on thelipase polypeptides and polynucleotides. The invention further relatesto procedures for producing the lipase polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

Lipases are indispensable for the bioconversion of lipids within anorganism through the catalysis of a variety of reactions that includehydrolysis, alcoholysis, acidolysis, esterfication and aminolysis. Inhumans, several lipases have been identified which possess lipolyticactivities that regulate levels of triglycerides and cholesterol in thebody. Enzymes from this superfamily, include lipoprotein lipase (LPL),hepatic lipase (HL), and pancreatic lipase (PL). While all three enzymeshydrolyze lipid emulsions and have similar aqueous-lipid interfacialcatalytic activities, they each possess unique properties andphysiological functions. All three enzymes act preferentially on thesn-1 and sn-3 bonds of triglycerides, to release fatty acids from theglycerol backbone (Dolphin et al. (1992) Structure and Function ofApolipoproteins, Rosseneu, M. (ed) CRC Press, Inc, Boca Ratan, 295-362).However, while PL completes the hydrolysis of alimentary triglycerides,the LPL and HL enzymes hydrolyze triglycerides found in circulatinglipoproteins.

Due to the insolubility of lipids in water, the plasma transportscomplex lipids among various tissues as components of lipoproteins. Eachlipoprotein contains a neutral lipid core composed of triacylglyceroland/or a cholesterol ester. Surrounding the core is a layer of proteins,phospholipids, and cholesterol. The proteins associated with thelipoprotein comprise a class of proteins referred to as apoproteins(apo). Based on apoprotein composition and density, lipoproteins havebeen classified into five major types that include chylomicrons,high-density lipoproteins (HDL), intermediate-density lipoproteins(IDL), low-density lipoproteins (LDL), and very-low density lipoproteins(VLDL).

Lipoprotein lipase (LPL) is the major enzyme responsible for thehydrolysis of triglyceride molecules present in circulatinglipoproteins. LPL is associated with the luminal side of capillaries andarteries through an interaction with heparin-sulfate chains ofproteoglycans and/or by glycerol phosphatidylinostintol. With the helpof the activator apo CII, LPL hydrolyzes triglycerides of lipoproteinsto produce free fatty acids. Muscle and adipose tissue assimilate thesefatty acids. Alternatively, the fatty acids can be bound to albumin andtransported to other tissues. As the lipase hydrolyzes the triglyceridesof the lipoprotein, the particles become smaller and are often referredto as lipoprotein remnants. Within the plasma compartment, LPL convertschylomicrons to remnants and begins the cascade requirements forconversion of VLDL to LDL.

In its active form, LPL is a glycosylated non-covalent homodimer, witheach subunit containing a binding site for heparin and apolipoprotein(apo) CII, an activator protein required for LPL activity. In additionto hydrolysis of triglycerides, LPL can hydrolyze a variety of othersubstrates, for example, long and short chain glycerides, phospholipidsand various synthetic substrates (Olivecrona et al. (1987) LipoproteinLipase Borensztajn, J. (ed) Evener Publisher, Inc., pages 15-58).

In addition to the lypolytic activity of LPL described above, LPL playsadditional roles in lipid metabolism. After sufficient hydrolysis,lipoprotein lipase is released from proteoglycans and travels with theremnants of the chylomicrons or VLDL. In the plasma LPL may then act tosequester the remnant particles on surface proteoglycans. SubsequentlyLPL can act as a ligand for receptors such as the LDL receptor,LDL-receptor related protein, gp330, or the VLDL receptor. Thisinteraction with the cell surface receptor facilitates the uptake anddegradation of plasma lipoproteins by cells (Williams et al. (1992) J.Biol. Chem 267:13284-13292 and Nykjaer et al. (1993) J. Biol. Chem.268:15048-15055).

Furthermore, LPL expressed in macrophages has been implicated in thecellular uptake of lipoprotein lipids and fat soluble vitamins, thedegradation of lipid-containing pathogens and cell debris, and thecreation of fatty acids for the energy requirements of the cell.

Disruption of LPL activity has also been implicated in other biologicalfunctions including, for example, enhanced oxidative stress in bloodcells, increased fluidity of the membrane components of these cells andincreases the susceptibility of their mitochondrial DNA to structuralalterations (Ven Murthy et al. (1996) Acta Biochimica Polonica43:227-40).

Hepatic lipase (HL) has functions in lipid metabolism similar to thoseof LPL. HL is located on the surface of liver sinusoids throughglycosaminoglycan links where it interacts with lipoproteins andhydrolyzes triglycerides into free fatty acids. Unlike LPL, the activityof HL does not require an activator, but its activity may be stimulatedby apo E. Thus, the preferred substrates of HL are the triglycerides ofapo E-containing lipoproteins, such as chylomicron remnants, IDL, andHDL. Furthermore, the actions of HL on HDL is important in the reversecholesterol transport process, a mechanism thought to reduce excessaccumulation of cholesterol in hepatic tissue.

Like LPL, hepatic lipase has also been implicated in the uptake anddegradation of lipoprotein in the hepatic tissue. Evidence suggests thatHL may interact with cell surface receptors, such as those describedabove, and direct hepatic cellular uptake of lipoproteins andlipoprotein remnants. (Chappell et al. (1998) Progress in Lipid Research37:363-422).

In its active form, HL exists as a monomer comprising both triglyceridelipase activity and phospholipase activity. As with LPL, treatment withheparin, results in the release of HL from the cell surfaces. Whileglycosylation plays an important role in secretion and affinity of LPL,it does not seem to be crucial for HL activity.

Pancreatic lipase (PL) is synthesized in acinar cells of the exocrinepancreas along with its protein activator, colipase. The pancreatic ducttransports glycosylated PL and colipase into the duodenum. PL does notbecome anchored to membrane surfaces like LPL or HL. Instead, the freemonomer of PL interacts with colipase which helps to anchor the PL tothe lipid-water interface where the enzyme completes the hydrolysis ofalimentary triglycerides.

In summary, lipases play a key role in lipid metabolism by regulatinglevels of cholesterol and triglycerides and therefore influence majormetabolic processes including effects on lipid and lipoproteinconcentrations, energy homeostasis, body weight, and bodycomposition-parameters. Each of these metabolic consequences has beenassociated with common diseases, such as, hypertriglyceridemia,atherosclerosis, obesity and various other disease states describedfurther below.

Accordingly, lipases are a major target for drug action and development.Thus, it is valuable to the field of pharmaceutical development toidentify and characterize previously unknown lipases. The presentinvention advances the state of the art by providing a previouslyunidentified human lipase enzyme.

SUMMARY OF THE INVENTION

It is an object of the invention to identify novel lipases.

It is a further object of the invention to provide novel lipasepolypeptides that are useful as reagents or targets in assays applicableto treatment and diagnosis of lipase-mediated or -related disorders,especially disorders mediated by or related to lipase enzymes.

It is a further object of the invention to provide polynucleotidescorresponding to the novel lipase polypeptides that are useful astargets and reagents in assays applicable to treatment and diagnosis oflipase or lipase-mediated or -related disorders and useful for producingnovel lipase polypeptides by recombinant methods.

A specific object of the invention is to identify compounds that act asagonists and antagonists and modulate the expression of the novellipase.

A further specific object of the invention is to provide compounds thatmodulate expression of the lipase for treatment and diagnosis of lipaseand lipase-related disorders.

The invention is thus based on the identification of a novel humanlipase. The amino acid sequence is shown in SEQ ID NO:1. The nucleotidesequence is shown in SEQ ID NO:2.

The invention provides isolated lipase polypeptides, including apolypeptide having the amino acid sequence shown in SEQ ID NO:1 or theamino acid sequence encoded by the cDNA deposited as ATCC Patent DepositNo. PTA-1915 on May 24, 2000 (“the deposited cDNA”).

The invention also provides isolated lipase nucleic acid moleculeshaving the sequence shown in SEQ ID NO:2 or in the deposited cDNA.

The invention also provides variant polypeptides having an amino acidsequence that is substantially homologous to the amino acid sequenceshown in SEQ ID NO:1 or encoded by the deposited cDNA.

The invention also provides variant nucleic acid sequences that aresubstantially homologous to the nucleotide sequence shown in SEQ ID NO:2or in the deposited cDNA.

The invention also provides fragments of the polypeptide shown in SEQ IDNO:1 and nucleotide sequence shown in SEQ ID NO:2, as well assubstantially homologous fragments of the polypeptide or nucleic acid.

The invention further provides nucleic acid constructs comprising thenucleic acid molecules described herein. In a preferred embodiment, thenucleic acid molecules of the invention are operatively linked to aregulatory sequence.

The invention also provides vectors and host cells for expressing thelipase nucleic acid molecules and polypeptides, and particularlyrecombinant vectors and host cells.

The invention also provides methods of making the vectors and host cellsand methods for using them to produce the lipase nucleic acid moleculesand polypeptides.

The invention also provides antibodies or antigen-binding fragmentsthereof that selectively bind the lipase polypeptides and fragments.

The invention also provides methods of screening for compounds thatmodulate expression or activity of the lipase polypeptides or nucleicacid (RNA or DNA).

The invention also provides a process for modulating lipase polypeptideor nucleic acid expression or activity, especially using the screenedcompounds. Modulation may be used to treat conditions related toaberrant activity or expression of the lipase polypeptides or nucleicacids or aberrant activity resulting in the alteredaccumulation/degradation of lipids.

The invention also provides assays for determining the activity of orthe presence or absence of the lipase polypeptides or nucleic acidmolecules in a biological sample, including for disease diagnosis.

The invention also provides assays for determining the presence of amutation in the polypeptides or nucleic acid molecules, including fordisease diagnosis.

In still a further embodiment, the invention provides a computerreadable means containing the nucleotide and/or amino acid sequences ofthe nucleic acids and polypeptides of the invention, respectively.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows the nucleotide sequence (SEQ ID NO:2) and thededuced amino acid sequence (SEQ ID NO:1) of the novel lipase.

FIG. 2 shows an analysis of the lipase amino acid sequence: αβturn andcoil regions; hydrophilicity; amphipathic regions; flexible regions;antigenic index; and surface probability plot.

FIG. 3 shows a hydrophobicity plot of the lipase (SEQ ID NO:1). Theanalysis predicted an 35 amino acid signal peptide sequence at the aminoterminus of the protein. Transmembrane segments of both the full lengthlipase and the mature lipase are also shown.

FIG. 4 shows an analysis of the lipase open reading frame for aminoacids corresponding to specific functional sites of SEQ ID NO:1. Proteinkinase C phosphorylation sites are found from about amino acid 63 to 65;from about amino acid 111 to 113; from about amino acid 252 to 254; fromabout amino acid 316 to 318. Casein kinase II phophorylation sites arefound from about amino acid 114 to 117; from amino acid 205 to 208; fromamino acid 284 to 287. N-myristoylation sites are found from about aminoacid from about 13 to 18, with the modified amino acid at position 13;from about amino acid 110 to 115, with the modified amino acid atposition 110; from about amino acid 146 to 151, with the modified aminoacid at position 146, from about amino acid 155 to 160, with themodified amino acid at position 155, from about amino acid 175 to 180,with the modified amino acid at position 175.

FIG. 5 shows expression of the lipase mRNA in various tissues and celltypes in culture. The expression data was derived from RT-PCR of variouscDNA libraries. The primers used were designed to amplify codingsequences.

FIG. 6 shows expression of the lipase mRNA in normal and malignantbreast, lung, liver and colon tissues. The liver metastases are derivedfrom malignant colonic tissue. The expression data was derived fromRT-PCR designed to amplify coding sequences.

FIG. 7 shows expression of the lipase mRNA in normal and malignantbreast, lung, liver and colon tissues. The liver metastases are derivedfrom malignant colonic tissue. The expression data was derived fromRT-PCR designed to amplify the unstranslated region of the lipase.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides

The invention is based on the identification of a novel human lipase.Specifically, an expressed sequence tag (EST) was selected based onhomology to lipase sequences. This EST was used to design primers basedon sequences that it contains and used to identify a cDNA from a brainlibrary. Positive clones were sequenced and the overlapping fragmentswere assembled. Analysis of the assembled sequence revealed that thecloned cDNA molecule encodes a lipase.

The invention thus relates to a novel lipase having the deduced aminoacid sequence shown in FIG. 1 (SEQ ID NO:1) or having the amino acidsequence encoded by the cDNA insert of the plasmid deposited withAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on May 24, 2000 and assigned Patent DepositNumber PTA-1915.

The deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms. Thedeposits are provided as a convenience to those of skill in the art andis not an admission that a deposit is required under 35 U.S.C. §112. Thedeposited sequences, as well as the polypeptides encoded by thesequences, are incorporated herein by reference and controls in theevent of any conflict, such as a sequencing error, with description inthis application.

“Lipase polypeptide” or “lipase protein” refers to the polypeptide inSEQ ID NO:1 or encoded by the deposited cDNA. The term “lipase protein”or “lipase polypeptide”, however, further includes the numerous variantsdescribed herein, as well as fragments derived from the full-lengthlipase and variants.

Tissues and/or cells in which the lipase is expressed include, but arenot limited to those shown in FIGS. 5, 6, and 7. Tissues in which thegene is highly expressed include liver, fetal liver, breast, brain,fetal kidney, and testis. Moderate expression occurs in prostate,skeletal muscle, colon, kidney, and thyroid. Lower positive expressionoccurs in heart, fetal heart, small intestine, spleen, lung, ovary,vein, aorta, placenta, osteoblasts, cervix, esophagus, thymus, tonsil,and lymph node. The lipase is also expressed in malignant breast, lung,and colon tissue and in liver metastases derived from malignant colonictissues. Hence, the lipase is relevant to disorders involving thetissues in which it is expressed.

The present invention thus provides an isolated or purified lipasepolypeptide and variants and fragments thereof.

Based on Clustal W sequence alignment, highest homology was shown tolipase 1 precursor (triacylglycerol lipase) from Psychrobacter immobilis(Acc. No. Q02104).

As used herein, a polypeptide is said to be “isolated” or “purified”when it is substantially free of cellular material when it is isolatedfrom recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can be joined to another polypeptide with which itis not normally associated in a cell and still be considered “isolated”or “purified.”

The lipase polypeptides can be purified to homogeneity. It isunderstood, however, that preparations in which the polypeptide is notpurified to homogeneity are useful and considered to contain an isolatedform of the polypeptide. The critical feature is that the preparationallows for the desired function of the polypeptide, even in the presenceof considerable amounts of other components. Thus, the inventionencompasses various degrees of purity.

In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the lipase having less than about 30%(by dry weight) other proteins (i.e., contaminating protein), less thanabout 20% other proteins, less than about 10% other proteins, or lessthan about 5% other proteins. When the polypeptide is recombinantlyproduced, it can also be substantially free of culture medium, i.e.,culture medium represents less than about 20%, less than about 10%, orless than about 5% of the volume of the protein preparation.

A lipase polypeptide is also considered to be isolated when it is partof a membrane preparation or is purified and then reconstituted withmembrane vesicles or liposomes.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the lipase polypeptide in which itis separated from chemical precursors or other chemicals that areinvolved in its synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of the polypeptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

In one embodiment, the lipase polypeptide comprises the amino acidsequence shown in SEQ ID NO:1 or the mature form of the polypeptide.However, the invention also encompasses sequence variants. Variantsinclude a substantially homologous protein encoded by the same geneticlocus in an organism, i.e., an allelic variant.

Variants also encompass proteins derived from other genetic loci in anorganism, but having substantial homology to the lipase of SEQ ID NO:1.Variants also include proteins substantially homologous to the lipasebut derived from another organism, i.e., an ortholog. Variants alsoinclude proteins that are substantially homologous to the lipase thatare produced by chemical synthesis. Variants also include proteins thatare substantially homologous to the lipase that are produced byrecombinant methods. It is understood, however, that variants excludeany amino acid sequences disclosed prior to the invention.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences are at leastabout 70-75%, typically at least about 80-85%, and most typically atleast about 90-95% or more homologous. A substantially homologous aminoacid sequence, according to the present invention, will be encoded by anucleic acid sequence hybridizing to the nucleic acid sequence, orportion thereof, of the sequence shown in SEQ ID NO:2 under stringentconditions as more fully described below.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (i.e., 100%=the entire coding sequence). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the lipase. Similarity is determinedby conserved amino acid substitution. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Conservative substitutions are likely to bephenotypically silent. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

A preferred, non-limiting example of such a mathematical algorithm isdescribed in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs (version 2.0) as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLAST) can be used. See www.ncbi.nlm.nih.gov. In one embodiment,parameters for sequence comparison can be set at score=100,wordlength=12, or can be varied (e.g., W=5 or W=20).

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman et al. (1970) (J. Mol. Biol.48:444-453) algorithm which has been incorporated into the GAP programin the GCG software package (available at www.gcg.com), using either aBLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (Devereux et al. (1984) Nucleic Acids Res. 12(1):387) (availableat www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the CGC sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Additional algorithms forsequence analysis are known in the art and include ADVANCE and ADAM asdescribed in Torellis et al. (1994) Comput. Appl. Biosci. 10:3-5; andFASTA described in Pearson et al. (1988) PNAS 85:2444-8.

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these.

Variant polypeptides can be fully functional or can lack function in oneor more activities. Thus, in the present case, variations can affect thefunction of the lipase at a variety of biological levels, including,disrupting interactions with the proteoglycans, such as CSPG, HSPG,DSPG, disrupting interaction with cell surface receptors, such as theLDL receptor, LDL-related receptor protein, gp330, or the VLDL receptor,disrupting interactions with heparin, disrupting interactions withapoproteins or lipoproteins, disrupting interactions with activatormolecules, such as apo CII or colipase, disrupting triglyceride lipaseactivity or phospholipase activity, or disrupting homodimer formation.Variant polypeptides having such defects have been identified for LPLand are described in, for example, Murthy et al. (1996) Pharmacol. Ther.70:101-135, incorporated herein by reference for teaching thesevariations.

Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar aminoacids, which results in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyaffect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

As indicated, variants can be naturally-occurring or can be made byrecombinant means or chemical synthesis to provide useful and novelcharacteristics for the lipase polypeptide. This includes preventingimmunogenicity from pharmaceutical formulations by preventing proteinaggregation.

Useful variations further include alteration of catalytic activity. Forexample, one embodiment involves a variation at the binding site thatresults in binding but not hydrolysis, or slower hydrolysis, of thetriglyceride or phospholipid. A further useful variation results in anincreased rate of hydrolysis of the triglycerides or phospholipids.Additional variations include altered affinity for co-activatorproteins, cell surface receptors, proteoglycans, heparin, triglycerides,phospholipids, lipoproteins or apoproteins. A further useful variationat the same site can result in higher or lower affinity for substrates.Useful variations also include changes that result in affinity to adifferent lipoprotein or lipoprotein remnant than that normallyrecognized. Other variations could result in altered recognition ofapoproteins thereby changing the preferred lipoproteins hydrolyzed bythe lipase. Further useful variations affect the ability of the lipaseto be induced by various activators, including, but not limited to,those disclosed herein. Specific variations include truncations in whicha catalytic domain or substrate binding domain is deleted. Thisvariation results in a decrease or loss of lipid hydrolytic activity orsubstrate binding. Another useful variation includes one that preventsglycosylation. Further useful variations provide a fusion protein inwhich one or more domains or subregions are operationally fused to oneor more domains or subregions from another lipase. Specifically, adomain or subregion can be introduced that provides a rescue function toan enzyme not normally having this function or for recognition of aspecific substrate wherein recognition is not available to the originalenzyme. Further variations could affect specific subunit interaction,particularly required for homodimerization or interaction with activatorproteins. Other variations would affect developmental, temporal, ortissue-specific expression. Other variations would affect theinteraction with cellular components, such as transcriptional regulatoryfactors.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al. (1985) Science 244:1081-1085). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity, such as the ability to hydrolyze triglycerides orphospholipids in vitro. Alternatively, in vitro activity may be measuredby the ability to interact with various molecules, including but notlimited to, heparin, proteoglycans, cell surface receptors,lipoproteins, apoproteins or activator proteins. Sites that are criticalfor binding or recognition can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al. (1992) J Mol. Biol. 224:899-904; de Vos et al.(1992) Science 255:306-312).

The assays for lipase enzyme activity are well known in the art and canbe found, for example, in Brun et al. (1989) Metabolism 38:1005-1009,Brunzell et al. (1992) Atherosclerosis IX, Stein (eds.) R&L CreativeCommunications Ltd., Tel Aviv 271-273, Peeva et al. (1992) Int. J. Obes.Relat. Metab. Disord. 16:737-744, Ma et al. (1991) N. Engl. J. Med. 324:1761-1766, Ma et al. (1992) J. Biol. Chem. 267: 1918-1923, Connelly etal. (1987) J. Clin. Invest. 80: 1597-1606, Huff et al. (1990) J. LipidRes. 31: 385-396, and Hixson et al. (1990) J. Lipid Res. 31: 545-548.These assays include measurements of triglyceride or lipoproteinconcentrations in the blood stream. For lipases associated withproteoglycans, plasma lipolytic activity may be determined followingheparin treatment. In this protocol, lipase activity is measured with asynthetic triglyceride substrate using plasma samples obtained followingheparin administration. Post-heparin plasma may also be used to measurethe lipase mass by immunoassay to determine if a catalytically defectivelipase enzyme is released into the plasma. Lipase activity can also bedetermined in s.c. biopsies of adipose tissue and through the detectionof lipase gene mutations. Additional assays include measuring lipaseactivation by the co-activator molecules.

Substantial homology can be to the entire nucleic acid or amino acidsequence or to fragments of these sequences.

The invention thus also includes polypeptide fragments of the lipase.Fragments can be derived from the amino acid sequence shown in SEQ IDNO:1. However, the invention also encompasses fragments of the variantsof the lipase as described herein.

The fragments to which the invention pertains, however, are not to beconstrued as encompassing fragments that may be disclosed prior to thepresent invention.

Accordingly, a fragment can comprise at least about 8, 13, 15, 20, 25,30, 35, 40, 45, 50 or more contiguous amino acids. Fragments can retainone or more of the biological activities of the protein, for example theability to bind to polyglycan, interact with cell surface receptors,interact with activator molecules, catalyze triglyceride hydrolysis, orretain phospholipase activity. Fragments can be used as an immunogen togenerate lipase antibodies.

Biologically active fragments (peptides which are, for example, 5, 7,10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acidsin length) can comprise a domain or motif, e.g., catalytic sites, signalpeptides, transmembrane segments, and sites for protein kinase Cphosphorylation, casein kinase II phosphorylation, and N-myristoylation.Additional domains include catalytic domains involved in triglyceridehydrolysis and phospholipase activity, heparin binding sites,cell-surface receptor binding sites, triglyceride binding sites, sitesimportant for homodimerization or activator interaction, and sitesimportant for carrying out the other functions of the lipase asdescribed herein.

Such domains or motifs can be identified by means of routinecomputerized homology searching procedures.

Fragments, for example, can extend in one or both directions from thefunctional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100amino acids. Further, fragments can include sub-fragments of thespecific domains mentioned above, which sub-fragments retain thefunction of the domain from which they are derived.

These regions can be identified by well-known methods involvingcomputerized homology analysis.

The invention also provides fragments with immunogenic properties. Thesecontain an epitope-bearing portion of the lipase and variants. Theseepitope-bearing peptides are useful to raise antibodies that bindspecifically to a lipase polypeptide or region or fragment. Thesepeptides can contain at least 8, at least 10, 13, 15, or between atleast about 16 to about 30 amino acids.

Non-limiting examples of antigenic polypeptides that can be used togenerate antibodies include but are not limited to peptides derived froman extracellular site. Regions having a high antigenicity index areshown in FIG. 2. However, intracellularly-made antibodies(“intrabodies”) are also encompassed, which would recognizeintracellular peptide regions.

The epitope-bearing lipase polypeptides may be produced by anyconventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci. USA82:5131-5135). Simultaneous multiple peptide synthesis is described inU.S. Pat. No. 4,631,211.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the lipase fragment and an additional region fused to thecarboxyl terminus of the fragment.

The invention thus provides chimeric or fusion proteins. These comprisea lipase peptide sequence operatively linked to a heterologous peptidehaving an amino acid sequence not substantially homologous to thelipase. “Operatively linked” indicates that the lipase peptide and theheterologous peptide are fused in-frame. The heterologous peptide can befused to the N-terminus or C-terminus of the lipase or can be internallylocated.

In one embodiment the fusion protein does not affect lipase function perse. For example, the fusion protein can be a GST-fusion protein in whichthe lipase sequences are fused to the N- or C-terminus of the GSTsequences. Other types of fusion proteins include, but are not limitedto, enzymatic fusion proteins, for example beta-galactosidase fusions,yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions. Suchfusion proteins, particularly poly-His fusions, can facilitate thepurification of a recombinant lipase protein. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence. Therefore, inanother embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus.

EP-A-O 464 533 discloses fusion proteins comprising various portions ofimmunoglobulin constant regions. The Fc is useful in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists (Bennett et al.(1995) J. Mol. Recog. 8:52-58 (1995) and Johanson et al. J. Biol. Chem.270:9459-9471). Thus, this invention also encompasses soluble fusionproteins containing a lipase polypeptide and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. For some uses it is desirable toremove the Fc after the fusion protein has been used for its intendedpurpose, for example when the fusion protein is to be used as antigenfor immunizations. In a particular embodiment, the Fc part can beremoved in a simple way by a cleavage sequence, which is alsoincorporated and can be cleaved with factor Xa.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.(1992) Current Protocols in Molecular Biology). Moreover, manyexpression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A lipase-encoding nucleic acid canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the lipase.

Another form of fusion protein is one that directly affects lipasefunctions. Accordingly, a lipase polypeptide is encompassed by thepresent invention in which one or more of the lipase domains (or partsthereof) has been replaced by homologous lipase domains (or partsthereof) from another species. Accordingly, various permutations arepossible. One or more functional sites as disclosed herein from thespecifically disclosed lipase can be replaced by one or more functionalsites from a corresponding lipase of another species. Thus, chimericlipases can be formed in which one or more of the native domains orsubregions has been replaced by another. For example, the catalyticdomain of the lipase of the present invention may be replaced by thecatalytic domain of a different lipase polypeptide. Alternatively,protein domains that mediate the interaction with lipoproteins ordomains that mediated the uptake of lipoproteins by cell surfacereceptors can be used to replace homologous domains of the lipase of thepresent invention. In doing so the binding affinity to varioussubstrates and/or the rate of catalysis is altered.

Additionally, chimeric lipase proteins can be produced in which one ormore functional sites is derived from a different member of the lipasesuperfamily. It is understood however that sites could be derived fromlipase families that occur in the mammalian genome but which have notyet been discovered or characterized. Such sites include but are notlimited to any of the functional sites disclosed herein.

The isolated lipase can be purified from any of the cells that naturallyexpress it, including, but not limited to those shown in FIGS. 5, 6, and7. Tissues in which the gene is highly expressed include liver, fetalliver, breast, brain, fetal kidney, and testis. Moderate expressionoccurs in prostate, skeletal muscle, colon, kidney, and thyroid. Lowerpositive expression occurs in heart, fetal heart, small intestine,spleen, lung, ovary, vein, aorta, placenta, osteoblasts, cervix,esophagus, thymus, tonsil, and lymph node. The lipase is also expressedin normal liver and in normal and malignant breast, lung, and colontissue and in liver metastases derived from malignant colonic tissues.Alternatively, the lipase may be purified from cells that have beenaltered to express it (recombinant), or synthesized using known proteinsynthesis methods.

In one embodiment, the protein is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the lipasepolypeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally-occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in polypeptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art.

Accordingly, the polypeptides also encompass derivatives or analogs inwhich a substituted amino acid residue is not one encoded by the geneticcode, in which a substituent group is included, in which the maturepolypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or in which the additional amino acids are fused to the maturepolypeptide, such as a leader or secretory sequence or a sequence forpurification of the mature polypeptide or a pro-protein sequence.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well-known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth.Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y. Acad. Sci.663:48-62).

As is also well known, polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of lipase, and theymay be circular, with or without branching, generally as a result ofpost-translation events, including natural processing events and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translational natural processes and by synthetic methods.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.Blockage of the amino or carboxyl group in a polypeptide, or both, by acovalent modification, is common in naturally-occurring and syntheticpolypeptides. For instance, the aminoterminal residue of polypeptidesmade in E. coli, prior to proteolytic processing, almost invariably willbe N-formylmethionine.

The modifications can be a function of how the protein is made. Forrecombinant polypeptides, for example, the modifications will bedetermined by the host cell posttranslational modification capacity andthe modification signals in the polypeptide amino acid sequence.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications.

The same type of modification may be present in the same or varyingdegree at several sites in a given polypeptide. Also, a givenpolypeptide may contain more than one type of modification.

Polypeptide Uses

The protein sequences of the present invention can be used as a “querysequence” to perform a search against public databases to, for example,identify other family members or related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) of Altschulet al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to the nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the proteins of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seewww.ncbi.nlm.nih.gov.

The lipase polypeptides are useful for producing antibodies specific forthe lipase protein, regions, or fragments. Regions having a highantigenicity index score are shown in FIG. 2.

The lipase polypeptides are useful for biological assays related tolipase function. Such assays involve any of the known functions oractivities or properties useful for diagnosis and treatment of lipase-or lipase-related conditions or conditions in which expression of thelipase is relevant, such as in hypertriacylglycerolaemia, obesity,atherogenesis, and the various other conditions described herein.Potential assays have been disclosed herein.

The lipase polypeptides are also useful in drug screening assays, incell-based or cell-free systems. Cell-based systems can be native, i.e.,cells that normally express the lipase, as a biopsy or expanded in cellculture. In one embodiment, however, cell-based assays involverecombinant host cells expressing the lipase.

Determining the ability of the test compound to interact with the lipasecan also comprise determining the ability of the test compound topreferentially bind to the polypeptide as compared to the ability of aknown binding molecule (e.g., an activator (such as colipase, apo CII),cell surface receptor, heparin, proteoglycan, triglyceride, orphospholipid, or lipoprotein) to bind to the polypeptide.

The polypeptides can be used to identify compounds that modulate lipaseactivity. Modulators of lipase activity comprise agents that influencethe enzyme at a variety of biological levels, including, but not limitedto agents that disrupt the interaction with the proteoglycans of thecell wall, such as HSPG-degrading enzymes, heparin, chlorate, or APOE;agents that disrupt the interaction with cell surface receptors; agentswhich disrupt the interaction with activator molecules or homodimerformation; agents that disrupt interaction with lipoproteins; or agentsthat disrupt triglyceride hydrolysis or phospholipase activity.

The tissue specific regulation of lipase is complex with identicalmodulators regulating activity differently under various metabolicconditions. While specific modulators of lipase activity have beendescribed above, additional modulators include, but are not limited to,apoproteins and a non-proteoglycan LPL-binding protein having sequencehomology to apo B and apo B (Sivaram et al. (1992) J. Biol. Chem.267:16517-16552; Sivaram et al. (1994) J. Biol. Chem. 269:9409-9412). Ithas also been postulated that the lipolysis-stimulated receptor (LSR)plays a role in LPL activation (Yen et al (1994) Biochemistry33:1172-1180). Additional modulators of lipase activity include,fasting, feeding, growth hormone, insulin, exercise, estrogen, thyroidhormone, catecholamines, hormones of the adrenergic system, vitamin Dderivatives, glucagon, catecholamines, glucocorticoids, and 1, 25dihydroxy-vitamin D. Further modulators comprise inflammatory mediatorssuch as cytokines, interleukins, and interferons.

Modulators associated with an increase activity of lipase activityinclude, but are not limited to various apoproteins, such as apo CII,and glycosylation. Furthermore, lipase enzymatic activity is stabilizedin the presence of lipids or by binding to lipid-water interfaces anddetergents, such as deoxycholate. Modulators associated with a decreasein lipase activity include, but are not limited to, increasedconcentrations of apo CII or apo CIII (Shirari et al. (1981) Biochim.Biophys. Acta 665:504-510), TNF (Kern et al. (1997) Journal of Nutrition127:1917S-1922S), fatty acids, high salt concentrations, and Orlistar(La Roche, Basele).

Both transcription and post-transcriptional levels of lipase expressionare regulated by various dietary, environmental, and developmentalfactors and include, for example, hormones, such as insulin, thyroidhormone, and glucocorticoids (Pykalisto et al. (1976) J. clin.Endocronol. Metab. 43:591-600; Nillson-Ehle et al. (1980) Annual RevBiochem 49:667-693; and Cryer et al. (1981) Int. J. Biochem 13:525-541).Various transcriptional factors such as CEBP, ADD-1, SREBP-1 and PPAR 6also regulate expression of specific lipases. It is understood,therefore, that such compounds can be identified not only by means ofdirect interaction with the lipase, but by means of any of thecomponents that functionally interact with the disclosed lipase. Thisincludes, but is not limited to, any of those components disclosedherein.

Both lipase and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the lipase. These compounds can be further screened against afunctional lipase to determine the effect of the compound on the lipaseactivity. Compounds can be identified that activate (agonist) orinactivate (antagonist) the lipase to a desired degree. Modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject).

The lipase polypeptides can be used to screen a compound for the abilityto stimulate or inhibit interaction between the lipase protein and atarget molecule that normally interacts with the lipase protein. Thetarget can be a lipoprotein, lipoprotein remnant, apoprotein, cellsurface receptors, heparin, proteoglycan, triglyceride, phospholipid oranother component of the pathway with which the lipase protein normallyinteracts. The assay includes the steps of combining the lipase proteinwith a candidate compound under conditions that allow the lipase proteinor fragment to interact with the target molecule, and to detect theformation of a complex between the lipase protein and the target or todetect the biochemical consequence of the interaction with the lipaseand the target. Any of the associated effects of triglyceride hydrolysisor phospholipase function can be assayed. This includes the productionof fatty acids from triglycerides and phospholipids.

Determining the ability of the lipase to bind to a target molecule canalso be accomplished using a technology such as real-time BimolecularInteraction Analysis (BIA). Sjolander et al. (1991) Anal. Chem.63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore™). Changes in the optical phenomenon surfaceplasmon resonance (SPR) can be used as an indication of real-timereactions between biological molecules.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, K. S. (1997)Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 3 7:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten (1992) Biotechniques13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor(1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409),spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc.Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990)Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla etal. (1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.Biol. 222:301-310); (Ladner supra).

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84;Houghten et al. (1991) Nature 354:84-86) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fall-length lipase or fragment thatcompetes for substrate binding. Other candidate compounds include mutantlipases or appropriate fragments containing mutations that affect lipasefunction and compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not hydrolyze the triglyceride orphospholipid, is encompassed by the invention.

Other candidate compounds include lipase protein or protein analog thatbinds to the lipid, lipoprotein, proteoglycan, cell surface receptor, orother substrates identified herein but is not released or releasedslowly. Other candidate compounds include analogs of the other naturalsubstrates, such as substrates that bind to but are not released orreleased more slowly. Further candidate compounds include activators ofthe lipases, including but not limited to, those disclosed herein.

The invention provides other end points to identify compounds thatmodulate (stimulate or inhibit) lipase activity. The assays typicallyinvolve an assay of events in the pathway that indicate lipase activity.This can include cellular events that are influenced by lipidmetabolism, such as but not limited to, lipid or lipoproteinconcentrations. Specific phenotypes include metabolic consequencesincluding effects on energy homeostasis, body weight and bodycomposition-parameters.

Assays are based on the multiple cellular functions of lipase enzymes.As described herein, these enzymes act at various levels in theregulation of lipid metabolism. Accordingly, assays can be based ondetection of any of the products produced by the lipase enzyme.

Further, the expression of genes that are up- or down-regulated byaction of the lipase can be assayed. In one embodiment, the regulatoryregion of such genes can be operably linked to a marker that is easilydetectable, such as luciferase.

Accordingly, any of the biological or biochemical functions mediated bythe lipase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art.

Binding and/or activating compounds can also be screened by usingchimeric lipase proteins in which one or more domains, sites, and thelike, as disclosed herein, or parts thereof, can be replaced by theirheterologous counterparts derived from other lipase protein. Forexample, a recognition or binding region can be used that interacts withdifferent substrate specificity and/or affinity than the native lipase.Accordingly, a different set of pathway components is available as anend-point assay for activation. Further, sites that are responsible fordevelopmental, temporal, or tissue specificity can be replaced byheterologous sites such that the lipase can be detected under conditionsof specific developmental, temporal, or tissue-specific expression.

The lipase polypeptides are also useful in competition binding assays inmethods designed to discover compounds that interact with the lipase.Thus, a compound is exposed to a lipase polypeptide under conditionsthat allow the compound to bind to or to otherwise interact with thepolypeptide. A lipase target, comprising a polypeptide or agent which isknown to interact with lipase, is also added to the mixture. If the testcompound interacts with the soluble lipase polypeptide, it decreases theamount of complex formed or the activity from the lipase target. Thistype of assay is particularly useful in cases in which compounds aresought that interact with specific regions of the lipase. Thus, thesoluble polypeptide that competes with the target lipase region isdesigned to contain peptide sequences corresponding to the region ofinterest.

Another type of competition-binding assay can be used to discovercompounds that interact with specific functional sites. As an example, acandidate compound can be added to a sample of the lipase. Compoundsthat interact with the lipase at the same site as a lipase substratedisclosed herein will reduce the amount of complex formed between thelipase and substrate. Accordingly, it is possible to discover a compoundthat specifically prevents interaction between the lipase and it varioussubstrates. A compound that competes with lipase catalytic activity willreduce the rate of triglyceride or phospholipid hydrolysis.Alternatively, a compound may also compete at the level of substrateinteraction. Accordingly, compounds can be discovered that directlyinteract with the lipase and interfere with its function. Such assayscan involve any other component that interacts with the lipase such asheparin, proteoglycans, lipoproteins, lipoprotein remnants, cell surfacereceptors, triglycerides, phospholipids, activator proteins, and othercompounds described herein.

To perform cell free drug screening assays, it is desirable toimmobilize either the lipase, or fragment, or its target molecule tofacilitate separation of complexes from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase/lipase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes is dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of lipase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a lipase-binding target component, such as, activator proteins, cellsurface receptors, lipoproteins, apoproteins, triglycerides orphospholipids and a candidate compound are incubated in thelipase-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the lipasetarget molecule, or which are reactive with lipase and compete with thetarget molecule; as well as enzyme-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Modulators of lipase activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedor affected by a lipase, by treating cells that express the lipase orcells in which lipase expression is desirable. These methods oftreatment include the steps of administering the modulators of lipaseactivity in a pharmaceutical composition as described herein, to asubject in need of such treatment.

Tissues and/or cells in which the lipase is expressed include, but arenot limited to those shown in FIGS. 5, 6, and 7. Tissues in which thegene is highly expressed include liver, fetal liver, breast, brain,fetal kidney, and testis. Moderate expression occurs in prostate,skeletal muscle, colon, kidney, and thyroid. Lower positive expressionoccurs in heart, fetal heart, small intestine, spleen, lung, ovary,vein, aorta, placenta, osteoblasts, cervix, esophagus, thymus, tonsil,and lymph node. The lipase is also expressed in malignant breast, lung,and colon tissue and in liver metastases derived from malignant colonictissues. Hence, the lipase is relevant to disorders involving thetissues in which it is expressed.

Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

Disorders involving the lung include, but are not limited to, congenitalanomalies; atelectasis; diseases of vascular origin, such as pulmonarycongestion and edema, including hemodynamic pulmonary edema and edemacaused by microvascular injury, adult respiratory distress syndrome(diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans—organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

Disorders involving the liver include, but are not limited to, hepaticinjury; jaundice and cholestasis, such as bilirubin and bile formation;hepatic failure and cirrhosis, such as cirrhosis, portal hypertension,including ascites, portosystemic shunts, and splenomegaly; infectiousdisorders, such as viral hepatitis, including hepatitis A-E infectionand infection by other hepatitis viruses, clinicopathologic syndromes,such as the carrier state, asymptomatic infection, acute viralhepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmunehepatitis; drug- and toxin-induced liver disease, such as alcoholicliver disease; inborn errors of metabolism and pediatric liver disease,such as hemochromatosis, Wilson disease, α₁-antitrypsin deficiency, andneonatal hepatitis; intrahepatic biliary tract disease, such assecondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

Disorders involving the brain include, but are not limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving the heart, include but are not limited to, heartfailure, including but not limited to, cardiac hypertrophy, left-sidedheart failure, and right-sided heart failure; ischemic heart disease,including but not limited to angina pectoris, myocardial infarction,chronic ischemic heart disease, and sudden cardiac death; hypertensiveheart disease, including but not limited to, systemic (left-sided)hypertensive heart disease and pulmonary (right-sided) hypertensiveheart disease; valvular heart disease, including but not limited to,valvular degeneration caused by calcification, such as calcific aorticstenosis, calcification of a congenitally bicuspid aortic valve, andmitral annular calcification, and myxomatous degeneration of the mitralvalve (mitral valve prolapse), rheumatic fever and rheumatic heartdisease, infective endocarditis, and noninfected vegetations, such asnonbacterial thrombotic endocarditis and endocarditis of systemic lupuserythematosus (Libman-Sacks disease), carcinoid heart disease, andcomplications of artificial valves; myocardial disease, including butnot limited to dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, and myocarditis; pericardial disease,including but not limited to, pericardial effusion and hemopericardiumand pericarditis, including acute pericarditis and healed pericarditis,and rheumatoid heart disease; neoplastic heart disease, including butnot limited to, primary cardiac tumors, such as myxoma, lipoma,papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effectsof noncardiac neoplasms; congenital heart disease, including but notlimited to, left-to-right shunts—late cyanosis, such as atrial septaldefect, ventricular septal defect, patent ductus arteriosus, andatrioventricular septal defect, right-to-left shunts—early cyanosis,such as tetralogy of fallot, transposition of great arteries, truncusarteriosus, tricuspid atresia, and total anomalous pulmonary venousconnection, obstructive congenital anomalies, such as coarctation ofaorta, pulmonary stenosis and atresia, and aortic stenosis and atresia,and disorders involving cardiac transplantation.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders of the breast include, but are not limited to, disorders ofdevelopment; inflammations, including but not limited to, acutemastitis, periductal mastitis, periductal mastitis (recurrent subareolarabscess, squamous metaplasia of lactiferous ducts), mammary ductectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

Disorders involving the testis and epididymis include, but are notlimited to, congenital anomalies such as cryptorchidism, regressivechanges such as atrophy, inflammations such as nonspecific epididymitisand orchitis, granulomatous (autoimmune) orchitis, and specificinflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sexcord-gonadal stroma including, but not limited to, leydig (interstitial)cell tumors and sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

Disorders involving the thyroid include, but are not limited to,hyperthyroidism; hypothyroidism including, but not limited to, cretinismand myxedema; thyroiditis including, but not limited to, hashimotothyroiditis, subacute (granulomatous) thyroiditis, and subacutelymphocytic (painless) thyroiditis; Graves disease; diffuse andmultinodular goiter including, but not limited to, diffuse nontoxic(simple) goiter and multinodular goiter; neoplasms of the thyroidincluding, but not limited to, adenomas, other benign tumors, andcarcinomas, which include, but are not limited to, papillary carcinoma,follicular carcinoma, medullary carcinoma, and anaplastic carcinoma; andcogenital anomalies.

Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

Disorders involving the small intestine include the malabsorptionsyndromes such as, celiac sprue, tropical sprue (postinfectious sprue),whipple disease, disaccharidase (lactase) deficiency,abetalipoproteinemia, and tumors of the small intestine includingadenomas and adenocarcinoma.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangaitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

Disorders involving the thymus include developmental disorders, such asDiGeorge syndrome with thymic hypoplasia or aplasia; thymic cysts;thymic hypoplasia, which involves the appearance of lymphoid follicleswithin the thymus, creating thymic follicular hyperplasia; and thymomas,including germ cell tumors, lynphomas, Hodgkin disease, and carcinoids.Thymomas can include benign or encapsulated thymoma, and malignantthymoma Type I (invasive thymoma) or Type II, designated thymiccarcinoma.

Disorders involving the ovary include, for example, polycystic ovariandisease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Bone-forming cells include the osteoprogenitor cells, osteoblasts, andosteocytes. The disorders of the bone are complex because they may havean impact on the skeleton during any of its stages of development.Hence, the disorders may have variable manifestations and may involveone, multiple or all bones of the body. Such disorders include,congenital malformations, achondroplasia and thanatophoric dwarfism,diseases associated with abnormal matix such as type 1 collagen disease,osteoporois, paget disease, rickets, osteomalacia, high-turnoverosteodystrophy, low-turnover of aplastic disease, osteonecrosis,pyogenic osteomyelitis, tuberculous osteomyelitism, osteoma, osteoidosteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous corticaldefects, fibrous dysplasia, fibrosarcoma, malignant fibroushistiocytoma, ewing saracoma, primitive neuroectodermal tumor, giantcell tumor, and metastatic tumors.

In addition, lipases influence a number of processes which affect thebiology of both blood vessel walls and the pancreas. Therefore, lipasesfind use in the treatment of disorders of blood vessels, which include,but are not limited to, responses of vascular cell walls to injury, suchas endothelial dysfunction and endothelial activation and intimalthickening; vascular diseases including, but not limited to, congenitalanomalies, such as arteriovenous fistula, atherosclerosis, andhypertensive vascular disease, such as hypertension; inflammatorydisease—the vasculitides, such as giant cell (temporal) arteritis,Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome(mucocutaneous lymph node syndrome), microscopic polyanglitis(microscopic polyarteritis, hypersensitivity or leukocytoclasticanglitis), Wegener granulomatosis, thromboanglitis obliterans (Buergerdisease), vasculitis associated with other disorders, and infectiousarteritis; Raynaud disease; aneurysms and dissection, such as abdominalaortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection(dissecting hematoma); disorders of veins and lymphatics, such asvaricose veins, thrombophlebitis and phlebothrombosis, obstruction ofsuperior vena cava (superior vena cava syndrome), obstruction ofinferior vena cava (inferior vena cava syndrome), and lymphangitis andlymphedema; tumors, including benign tumors and tumor-like conditions,such as hemangioma, lymphangioma, glomus tumor (glomangioma), vascularectasias, and bacillary angiomatosis, and intermediate-grade (borderlinelow-grade malignant) tumors, such as Kaposi sarcoma andhemangloendothelioma, and malignant tumors, such as angiosarcoma andhemangiopericytoma; and pathology of therapeutic interventions invascular disease, such as balloon angioplasty and related techniques andvascular replacement, such as coronary artery bypass graft surgery.

Disorders involving the pancreas include those of the exocrine pancreassuch as congenital anomalies, including but not limited to, ectopicpancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas; and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

Lipases play critical roles in lipid metabolism and are associated withvarious lipid-related pathologies in humans such as, but not limited to,Wolman's disease, hpertension, Type II diabetes, retinopathy andcholesterol ester storage disease. Furthermore, a decrease in LPLactivity impairs the catabolism of chylomicrons and VLDL resulting inmassive hypertriglyceridemia. Decreased LPL activity has been alsoassociated with many disorders, including for example, chylomicronemiasyndrome. This syndrome has multiple clinical symptoms andmanifestations review by Murthy et al. (1996) Pharmacol. Ther.70:101-135. Additional disorders resulting from defective LPL activityinclude, familial lipoprotein lipase deficiency with fastingchylomicronemia (type I hyperlipidemia) (Santamarina et al. (1992) CurrOpin Lipidology 3:186), LPL deficiency, familial combinedhyperlipidaemia (FCHL) (Babirak et al. (1992) Arteriosclerosis thromb.12:1176; Seed et al. (1994) Clin Invest 72: 100), hypertriglyceridemia,pancreatitis and abnormalities in post prandial lipemia. In addition,LPL activity is abnormally regulated in obesity (Kern et al. (1997) J.Nut. 127: 1917S-1922S) and is also affected by alcohol and severalhormones (Taskinen et al. (1987) Lipoprotein Lipase, Borensztajn J. (ed)Evener Chicago). Furthermore, changes in circulating lipoprotein andcreation of lipolytic products have been implicated in a number ofprocesses that affect the biology of vessel walls. For example,atherogenesis is associated with increased LPL activity. In addition,autoantibodies against LPL have been reported in patients withidiopathic thrombocytopenic purpura and Grave's disease (Kihara et al.(1989) N. Engl. J. Med. 320:1255-1259) and heparin resistance was notedin a case of disseminated lupus erythematosus (Glueck et al. (1969) Am.J. Med. 47:318-324). Polymorphisms in LDL gene have also been associatedwith altered levels of total and HDL cholesterol (Mitchell et al. (1994)Hum. Biol. 66:383-397), coronary heart disease (Mattu et al. (1994)Arterioscler. Thromb. 14:1090-1097), and insulin resistance (Cole et al.(1993) Genet. Epidemiol. 10:177-188).

The hydrolysis of HDL by hepatic lipase regulates cholesterol levels inhepatic tissue. Pathologies associated with cholesterol include, but arenot limited to, atherosclerosis, xanthomas, inflammation and necrosis,cholesterolosis and gall stone formation.

The lipase polypeptides are thus useful for treating a lipase-associateddisorder characterized by aberrant expression or activity of a lipase.The polypeptides can also be useful for treating a disordercharacterized by excessive amounts of lipoproteins, triglycerides orcholesterol. In one embodiment, the method involves administering anagent (e.g., an agent identified by a screening assay described herein),or combination of agents that modulates (e.g., upregulates ordownregulates) expression or activity of the protein. In anotherembodiment, the method involves administering the lipase as therapy tocompensate for reduced or aberrant expression or activity of theprotein. In another embodiment, the lipase polypeptides are useful fortreating breast, lung, colon, and liver cancers.

Methods for treatment include but are not limited to the use of solublelipase or fragments of the lipase protein that compete for substratesincluding those disclosed herein. These lipases or fragments can have ahigher affinity for the target so as to provide effective competition.

Stimulation of activity is desirable in situations in which the proteinis abnormally downregulated and/or in which increased activity is likelyto have a beneficial effect. Likewise, inhibition of activity isdesirable in situations in which the protein is abnormally upregulatedand/or in which decreased activity is likely to have a beneficialeffect. In one example of such a situation, a subject has a disordercharacterized by aberrant metabolism of lipids resulting in alteredlipoprotein concentrations, energy homeostasis, body weight,artherosclerosis, and body weight parameters.

In yet another aspect of the invention, the proteins of the inventioncan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO 94/10300), to identify other proteins(captured proteins) which bind to or interact with the proteins of theinvention and modulate their activity.

The lipase polypeptides also are useful to provide a target fordiagnosing a disease or predisposition to disease mediated by thelipase, including, but not limited to, diseases involving tissues inwhich the lipase are expressed as disclosed herein. Accordingly, methodsare provided for detecting the presence, or levels of, the lipase in acell, tissue, or organism. The method involves contacting a biologicalsample with a compound capable of interacting with the lipase such thatthe interaction can be detected.

The polypeptides are also useful for treating a disorder characterizedby reduced amounts of these components. Thus, increasing or decreasingthe activity of the lipase is beneficial to treatment. The polypeptidesare also useful to provide a target for diagnosing a diseasecharacterized by excessive substrate or reduced levels of substrate.Accordingly, where substrate is excessive, use of the lipasepolypeptides can provide a diagnostic assay. Furthermore, for example,lipases having reduced activity can be used to diagnose conditions inwhich reduced substrate is responsible for the disorder.

One agent for detecting lipase is an antibody capable of selectivelybinding to the lipase polypeptide. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The lipase also provides a target for diagnosing active disease, orpredisposition to disease, in a patient having a variant lipase. Thus,lipase can be isolated from a biological sample and assayed for thepresence of a genetic mutation that results in an aberrant protein. Thisincludes amino acid substitution, deletion, insertion, rearrangement,(as the result of aberrant splicing events), and inappropriatepost-translational modification. Analytic methods include alteredelectrophoretic mobility, altered tryptic peptide digest, altered lipaseactivity in cell-based or cell-free assay, alteration in binding to orhydrolysis of lipids, binding to activator proteins, cell surfacereceptors, apoproteins, lipoproteins, proteoglycans, heparin, orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein in general or in a lipase specifically,including assays discussed herein.

In vitro techniques for detection of lipase include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. Alternatively, the protein can be detected in vivoin a subject by introducing into the subject a labeled anti-lipaseantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques. Particularly useful are methods, whichdetect the allelic variant of the lipase expressed in a subject, andmethods, which detect fragments of the lipase in a sample.

The lipase polypeptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985, and Linder, M. W. (1997) Clin.Chem. 43(2):254-266. The clinical outcomes of these variations result insevere toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes affects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the lipase in which one or more ofthe lipase functions in one population is different from those inanother population. The polypeptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in alipase-based treatment, polymorphism may give rise to catalytic regionsthat are more or less active. Accordingly, dosage would necessarily bemodified to maximize the therapeutic effect within a given populationcontaining the polymorphism. As an alternative to genotyping, specificpolymorphic polypeptides could be identified.

The lipase polypeptides are also useful for monitoring therapeuticeffects during clinical trials and other treatment. Thus, thetherapeutic effectiveness of an agent that is designed to increase ordecrease gene expression, protein levels or lipase activity can bemonitored over the course of treatment using the lipase polypeptides asan end-point target. The monitoring can be, for example, as follows: (i)obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression oractivity of the protein in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the protein in thepost-administration samples; (v) comparing the level of expression oractivity of the protein in the pre-administration sample with theprotein in the post-administration sample or samples; and (vi)increasing or decreasing the administration of the agent to the subjectaccordingly.

Antibodies

The invention also provides antibodies that selectively bind to thelipase and its variants and fragments. An antibody is considered toselectively bind, even if it also binds to other proteins that are notsubstantially homologous with the lipase. These other proteins sharehomology with a fragment or domain of the lipase polypeptide. Thisconservation in specific regions gives rise to antibodies that bind toboth proteins by virtue of the homologous sequence. In this case, itwould be understood that antibody binding to the lipase is stillselective.

To generate antibodies, an isolated lipase polypeptide is used as animmunogen to generate antibodies using standard techniques forpolyclonal and monoclonal antibody preparation. Either the full-lengthprotein or antigenic peptide fragment can be used. Regions having a highantigenicity index are shown in FIG. 2.

Antibodies are preferably prepared from these regions or from discretefragments in these regions. However, antibodies can be prepared from anyregion of the peptide as described herein. A preferred fragment producesan antibody that diminishes or completely prevents substrate hydrolysisor binding. Antibodies can be developed against the entire lipaseprotein or domains of the lipase as described herein. Antibodies canalso be developed against specific functional sites as disclosed herein.

The antigenic peptide can comprise a contiguous sequence of at least 8,13, 14, 15, or 30 amino acid residues. In one embodiment, fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions. These fragments are not to be construed,however, as encompassing any fragments, which may be disclosed prior tothe invention.

Antibodies can be polyclonal or monoclonal. An intact antibody, or afragment thereof (e.g. Fab or F(ab′)₂) can be used.

Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

An appropriate immunogenic preparation can be derived from native,recombinantly expressed, or chemically synthesized peptides.

Antibody Uses

The antibodies can be used to isolate a lipase by standard techniques,such as affinity chromatography or immunoprecipitation. The antibodiescan facilitate the purification of the natural lipase from cells andrecombinantly produced lipase expressed in host cells.

The antibodies are useful to detect the presence of lipase in cells ortissues to determine the pattern of expression of the lipase amongvarious tissues in an organism and over the course of normaldevelopment.

The antibodies can be used to detect lipase in situ, in vitro, or in acell lysate or supernatant in order to evaluate the abundance andpattern of expression.

The antibodies can be used to assess abnormal tissue distribution orabnormal expression during development.

Antibody detection of circulating fragments of the full length lipasecan be used to identify lipase turnover.

Further, the antibodies can be used to assess lipase expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to lipidmetabolism. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, or level of expression of thelipase protein, the antibody can be prepared against the normal lipaseprotein. If a disorder is characterized by a specific mutation in thelipase, antibodies specific for this mutant protein can be used to assayfor the presence of the specific mutant lipase polypeptides. However,intracellularly-made antibodies (“intrabodies”) are also encompassed,which would recognize intracellular lipase-peptide regions.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Antibodies can be developed against the whole lipase or portions of thelipase.

The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting lipase expression level or the presenceof aberrant lipase proteins and aberrant tissue distribution ordevelopmental expression, antibodies directed against the lipase orrelevant fragments can be used to monitor therapeutic efficacy.

Antibodies accordingly can be used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic lipases can be used to identifyindividuals that require modified treatment modalities.

The antibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant lipase analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

The antibodies are also useful for tissue typing. Thus, where a specificlipase has been correlated with expression in a specific tissue,antibodies that are specific for this lipase can be used to identify atissue type.

The antibodies are also useful in forensic identification. Accordingly,where an individual has been correlated with a specific geneticpolymorphism resulting in a specific polymorphic protein, an antibodyspecific for the polymorphic protein can be used as an aid inidentification.

The antibodies are also useful for inhibiting the various lipasefunctions as described herein.

These uses can also be applied in a therapeutic context in whichtreatment involves inhibiting lipase function. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact lipase associated with a cell.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. For an overview of this technology forproducing human antibodies, see Lonberg et al. (1995) Int. Rev. Immunol.13:65-93. For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No.5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S.Pat. No. 5,545,806.

The invention also encompasses kits for using antibodies to detect thepresence of a lipase protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting lipase in a biological sample; means fordetermining the amount of lipase in the sample; and means for comparingthe amount of lipase in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect lipase.

Polynucleotides

The nucleotide sequence in SEQ ID NO:2 was obtained by sequencing thedeposited human cDNA. Accordingly, the sequence of the deposited cloneis controlling as to any discrepancies between the two and any referenceto the sequence of SEQ ID NO:2 includes reference to the sequence of thedeposited cDNA.

The specifically disclosed cDNA comprises the coding region and 5′ and3′ untranslated sequences in SEQ ID NO:2.

The invention provides isolated polynucleotides encoding the novellipase. The term “lipase polynucleotide” or “lipase nucleic acid” refersto the sequence shown in SEQ ID NO:2 or in the deposited cDNA. The term“lipase polynucleotide” or “lipase nucleic acid” further includesvariants and fragments of the lipase polynucleotide.

An “isolated” lipase nucleic acid is one that is separated from othernucleic acid present in the natural source of the lipase nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the lipase nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. However, there can be someflanking nucleotide sequences, for example up to about 5 KB. Theimportant point is that the lipase nucleic acid is isolated fromflanking sequences such that it can be subjected to the specificmanipulations described herein, such as recombinant expression,preparation of probes and primers, and other uses specific to the lipasenucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a cDNA or RNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

In some instances, the isolated material will form part of a composition(for example, a crude extract containing other substances), buffersystem or reagent mix. In other circumstances, the material may bepurified to essential homogeneity, for example as determined by PAGE orcolumn chromatography such as HPLC. Preferably, an isolated nucleic acidcomprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

In some instances, the isolated material will form part of a composition(or example, a crude extract containing other substances), buffer systemor reagent mix. In other circumstances, the material may be purified toessential homogeneity, for example as determined by PAGE or columnchromatography such as HPLC. Preferably, an isolated nucleic acidcomprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

The lipase polynucleotides can encode the mature protein plus additionalamino or carboxyterminal amino acids, or amino acids interior to themature polypeptide (when the mature form has more than one polypeptidechain, for instance). Such sequences may play a role in processing of aprotein from precursor to a mature form, facilitate protein trafficking,prolong or shorten protein half-life or facilitate manipulation of aprotein for assay or production, among other things. As generally is thecase in situ, the additional amino acids may be processed away from themature protein by cellular enzymes.

The lipase polynucleotides include, but are not limited to, the sequenceencoding the mature polypeptide alone, the sequence encoding the maturepolypeptide and additional coding sequences, such as a leader orsecretory sequence (e.g., a pre-pro or pro-protein sequence), thesequence encoding the mature polypeptide, with or without the additionalcoding sequences, plus additional non-coding sequences, for exampleintrons and non-coding 5′ and 3′ sequences such as transcribed butnon-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the polynucleotide may befused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Lipase polynucleotides can be in the form of RNA, such as mRNA, or inthe form DNA, including cDNA and genomic DNA obtained by cloning orproduced by chemical synthetic techniques or by a combination thereofThe nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

Lipase nucleic acid can comprise the nucleotide sequence shown in SEQ IDNO:2, corresponding to human cDNA.

In one embodiment, the lipase nucleic acid comprises only the codingregion.

The invention further provides variant lipase polynucleotides, andfragments thereof, that differ from the nucleotide sequence shown in SEQID NO:2 due to degeneracy of the genetic code and thus encode the sameprotein as that encoded by the nucleotide sequence shown in SEQ ID NO:2.

The invention also provides lipase nucleic acid molecules encoding thevariant polypeptides described herein. Such polynucleotides may benaturally occurring, such as allelic variants (same locus), homologs(different locus), and orthologs (different organism), or may beconstructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to polynucleotides, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions.

Typically, variants have a substantial identity with a nucleic acidmolecule of SEQ ID NO:2 and the complements thereof. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a lipase that is at least about 60-65%, 65-70%,typically at least about 70-75%, more typically at least about 80-85%,and most typically at least about 90-95% or more homologous to thenucleotide sequence shown in SEQ ID NO:2. Such nucleic acid moleculescan readily be identified as being able to hybridize under stringentconditions, to the nucleotide sequence shown in SEQ ID NO:2 or afragment of the sequence. It is understood that stringent hybridizationdoes not indicate substantial homology where it is due to generalhomology, such as poly A sequences, or sequences common to all or mostproteins or all lipase enzymes. Moreover, it is understood that variantsdo not include any of the nucleic acid sequences that may have beendisclosed prior to the invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a polypeptide at least about 60-65%homologous to each other typically remain hybridized to each other. Theconditions can be such that sequences at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 90%, atleast about 95% or more identical to each other remain hybridized to oneanother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. One exampleof stringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. In another non-limitingexample, nucleic acid molecules are allowed to hybridize in 6×sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morelow stringency washes in 0.2×SSC/0.1% SDS at room temperature, or by oneor more moderate stringency washes in 0.2×SSC/0.1% SDS at 42° C., orwashed in 0.2×SSC/0.1% SDS at 65° C. for high stringency. In oneembodiment, an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:1 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

As understood by those of ordinary skill, the exact conditions can bedetermined empirically and depend on ionic strength, temperature and theconcentration of destabilizing agents such as formamide or denaturingagents such as SDS. Other factors considered in determining the desiredhybridization conditions include the length of the nucleic acidsequences, base composition, percent mismatch between the hybridizingsequences and the frequency of occurrence of subsets of the sequenceswithin other non-identical sequences. Thus, equivalent conditions can bedetermined by varying one or more of these parameters while maintaininga similar degree of identity or similarity between the two nucleic acidmolecules.

The present invention also provides isolated nucleic acids that containa single or double stranded fragment or portion that hybridizes understringent conditions to the nucleotide sequence of SEQ ID NO:2 or thecomplement of SEQ ID NO:2. In one embodiment, the nucleic acid consistsof a portion of the nucleotide sequence of SEQ ID NO:2 or the complementof SEQ ID NO:2.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if afragment is disclosed prior to the present invention, that fragment isnot intended to be encompassed by the invention. When a sequence is notdisclosed prior to the present invention, an isolated nucleic acidfragment is at least about 6, preferably at least about 10, 13,18, 20,23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500 or morenucleotides in length. Nucleotide sequences from about 1517 to 1964 arenot disclosed prior to the invention. Longer fragments, for example, 30or more nucleotides in length, which encode antigenic proteins orpolypeptides described herein are useful.

Furthermore, the invention provides polynucleotides that comprise afragment of the full-length lipase polynucleotides. The fragment can besingle or double-stranded and can comprise DNA or RNA. The fragment canbe derived from either the coding or the non-coding sequence.

In another embodiment an isolated lipase nucleic acid encodes the entirecoding region. Other fragments include nucleotide sequences encoding theamino acid fragments described herein.

Thus, lipase nucleic acid fragments further include sequencescorresponding to the domains described herein, subregions alsodescribed, and specific functional sites. Lipase nucleic acid fragmentsalso include combinations of the domains, segments, and other functionalsites described above. A person of ordinary skill in the art would beaware of the many permutations that are possible.

Where the location of the domains or sites have been predicted bycomputer analysis, one of ordinary sill would appreciate that the aminoacid residues constituting these domains can vary depending on thecriteria used to define the domains.

However, it is understood that a lipase fragment includes any nucleicacid sequence that does not include the entire gene.

The invention also provides lipase nucleic acid fragments that encodeepitope bearing regions of the lipase proteins described herein.

Nucleic acid fragments, according to the present invention, are not tobe construed as encompassing those fragments that may have beendisclosed prior to the invention.

Polynucleotide Uses

The nucleotide sequences of the present invention can be used as a“query sequence” to perform a search against public databases, forexample, to identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25(1 7):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The nucleic acid fragments of the invention provide probes or primers inassays such as those described below. “Probes” are oligonucleotides thathybridize in a base-specific manner to a complementary strand of nucleicacid. Such probes include polypeptide nucleic acids, as described inNielsen et al. (1991) Science 254:1497-1500. Typically, a probecomprises a region of nucleotide sequence that hybridizes under highlystringent conditions to at least about 15, typically about 20-25, andmore typically about 40, 50 or 75 consecutive nucleotides of the nucleicacid sequence shown in SEQ ID NO:2 and the complements thereof. Moretypically, the probe further comprises a label, e.g., radioisotope,fluorescent compound, enzyme, or enzyme co-factor.

As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis using well-known methods (e.g., PCR, LCR) including, butnot limited to those described herein. The appropriate length of theprimer depends on the particular use, but typically ranges from about 15to 30 nucleotides. The term “primer site” refers to the area of thetarget DNA to which a primer hybridizes. The term “primer pair” refersto a set of primers including a 5′ (upstream) primer that hybridizeswith the 5′ end of the nucleic acid sequence to be amplified and a 3′(downstream) primer that hybridizes with the complement of the sequenceto be amplified.

The lipase polynucleotides are thus useful for probes, primers, and inbiological assays.

Where the polynucleotides are used to assess lipase properties orfunctions, such as in the assays described herein, all or less than allof the entire cDNA can be useful. Assays specifically directed to lipasefunctions, such as assessing agonist or antagonist activity, encompassthe use of known fragments. Further, diagnostic methods for assessinglipase function can also be practiced with any fragment, including thosefragments that may have been known prior to the invention. Similarly, inmethods involving treatment of lipase dysfunction, all fragments areencompassed including those, which may have been known in the art.

The lipase polynucleotides are useful as a hybridization probe for cDNAand genomic DNA to isolate a full-length cDNA and genomic clonesencoding the polypeptide described in SEQ ID NO:1 and to isolate cDNAand genomic clones that correspond to variants producing the samepolypeptide shown in SEQ ID NO:1 or the other variants described herein.Variants can be isolated from the same tissue and organism from whichthe polypeptides shown in SEQ ID NO:1 were isolated, different tissuesfrom the same organism, or from different organisms. This method isuseful for isolating genes and cDNA that are developmentally-controlledand therefore may be expressed in the same tissue or different tissuesat different points in the development of an organism.

The probe can correspond to any sequence along the entire length of thegene encoding the lipase. Accordingly, it could be derived from 5′noncoding regions, the coding region, and 3′ noncoding regions.

The nucleic acid probe can be, for example, the full-length cDNA of SEQID NO:2 or a fragment thereof that is sufficient to specificallyhybridize under stringent conditions to mRNA or DNA.

Fragments of the polynucleotides described herein are also useful tosynthesize larger fragments or full-length polynucleotides describedherein. For example, a fragment can be hybridized to any portion of anmRNA and a larger or full-length cDNA can be produced.

The fragments are also useful to synthesize antisense molecules ofdesired length and sequence.

Antisense nucleic acids of the invention can be designed using thenucleotide sequence of SEQ ID NO:2, and constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

Additionally, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670. PNAs can be further modified, e.g., to enhance theirstability, specificity or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. The synthesis of PNA-DNA chimeras can be performed as described inHyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res.24(17):3357-63, Mag et al. (1989) Nucleic Acids Res. 17:5973, andPeterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

The nucleic acid molecules and fragments of the invention can alsoinclude other appended groups such as peptides (e.g., for targeting hostcell lipases in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/0918) or the blood brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).

The lipase polynucleotides are also useful as primers for PCR to amplifyany given region of a lipase polynucleotide.

The lipase polynucleotides are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the lipase polypeptides. Vectors also include insertionvectors, used to integrate into another polynucleotide sequence, such asinto the cellular genome, to alter in situ expression of lipase genesand gene products. For example, an endogenous lipase coding sequence canbe replaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The lipase polynucleotides are also useful for expressing antigenicportions of the lipase proteins.

The lipase polynucleotides are also useful as probes for determining thechromosomal positions of the lipase polynucleotides by means of in situhybridization methods, such as FISH. (For a review of this technique,see Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques(Pergamon Press, New York), and PCR mapping of somatic cell hybrids. Themapping of the sequences to chromosomes is an important first step incorrelating these sequences with genes associated with disease.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland et al. (1987) Nature325:783-787).

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with a specified gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations, that are visible from chromosome spreads, or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

The lipase polynucleotide probes are also useful to determine patternsof the presence of the gene encoding the lipase and their variants withrespect to tissue distribution, for example, whether gene duplicationhas occurred and whether the duplication occurs in all or only a subsetof tissues. The genes can be naturally occurring or can have beenintroduced into a cell, tissue, or organism exogenously.

The lipase polynucleotides are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from genesencoding the polynucleotides described herein.

The lipase polynucleotides are also useful for constructing host cellsexpressing a part, or all, of the lipase polynucleotides andpolypeptides.

The lipase polynucleotides are also useful for constructing transgenicanimals expressing all, or a part, of the lipase polynucleotides andpolypeptides.

The lipase polynucleotides are also useful for making vectors thatexpress part, or all, of the lipase polypeptides.

The lipase polynucleotides are also useful as hybridization probes fordetermining the level of lipase nucleic acid expression. Accordingly,the probes can be used to detect the presence of, or to determine levelsof, lipase nucleic acid in cells, tissues, and in organisms. The nucleicacid whose level is determined can be DNA or RNA. Accordingly, probescorresponding to the polypeptides described herein can be used to assessgene copy number in a given cell, tissue, or organism. This isparticularly relevant in cases in which there has been an amplificationof the lipase genes.

Alternatively, the probe can be used in an in situ hybridization contextto assess the position of extra copies of the lipase genes, as onextrachromosomal elements or as integrated into chromosomes in which thelipase gene is not normally found, for example as a homogeneouslystaining region.

These uses are relevant for diagnosis of disorders involving an increaseor decrease in lipase expression relative to normal, such as adevelopmental or a metabolic disorder.

Tissues and/or cells in which the lipase is expressed include, but arenot limited to those shown in FIGS. 5, 6, and 7. Such tissues/cellsinclude liver, fetal liver, breast, brain, fetal kidney, and testis.Moderate expression occurs in prostate, skeletal muscle, colon, kidney,and thyroid. Lower positive expression occurs in heart, fetal heart,small intestine, spleen, lung, ovary, vein, aorta, placenta,osteoblasts, cervix, esophagus, thymus, tonsil, and lymph node. Thelipase is also expressed in malignant breast, lung, and colon tissue andin liver metastases derived from malignant colonic tissues. Hence, thelipase is relevant to disorders involving the tissues in which it isexpressed. As such, the gene is particularly relevant for the treatmentof disorders involving breast, lung, liver, and colon cancer. Disordersin which the lipase expression is relevant include, but are not limitedto those disclosed herein above.

Thus, the present invention provides a method for identifying a diseaseor disorder associated with aberrant expression or activity of lipasenucleic acid, in which a test sample is obtained from a subject andnucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presenceof the nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of the nucleic acid.

One aspect of the invention relates to diagnostic assays for determiningnucleic acid expression as well as activity in the context of abiological sample (e.g., blood, serum, cells, tissue) to determinewhether an individual has a disease or disorder, or is at risk ofdeveloping a disease or disorder, associated with aberrant nucleic acidexpression or activity. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated withexpression or activity of the nucleic acid molecules.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express the lipase, such as by measuring the levelof a lipase-encoding nucleic acid in a sample of cells from a subjecte.g., mRNA or genomic DNA, or determining if the lipase gene has beenmutated.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate lipase nucleic acid expression (e.g., antisense,polypeptides, peptidomimetics, small molecules or other drugs). A cellis contacted with a candidate compound and the expression of mRNAdetermined. The level of expression of the mRNA in the presence of thecandidate compound is compared to the level of expression of the mRNA inthe absence of the candidate compound. The candidate compound can thenbe identified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. The modulator can bind to the nucleicacid or indirectly modulate expression, such as by interacting withother cellular components that affect nucleic acid expression.

Modulatory methods can be performed in vitro (e.g., by culturing thecell with the agent) or, alternatively, in vivo (e.g., by administeringthe gent to a subject) in patients or in transgenic animals.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe lipase gene. The method typically includes assaying the ability ofthe compound to modulate the expression of the lipase nucleic acid andthus identifying a compound that can be used to treat a disordercharacterized by undesired lipase nucleic acid expression.

The assays can be performed in cell-based and cell-free systems.Cell-based assays include cells naturally expressing the lipase nucleicacid or recombinant cells genetically engineered to express specificnucleic acid sequences.

Alternatively, candidate compounds can be assayed in vivo in patients orin transgenic animals.

The assay for lipase nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the pathway. Further, the expression of genes that are up-or down-regulated in response to the lipase activity can also beassayed. In this embodiment the regulatory regions of these genes can beoperably linked to a reporter gene such as luciferase.

Thus, modulators of lipase gene expression can be identified in a methodwherein a cell is contacted with a candidate compound and the expressionof mRNA determined. The level of expression of lipase mRNA in thepresence of the candidate compound is compared to the level ofexpression of lipase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

Accordingly, the invention provides methods of treatment, with thenucleic acid as a target, using a compound identified through dragscreening as a gene modulator to modulate lipase nucleic acidexpression. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) oreffects on nucleic acid activity (e.g. when nucleic acid is mutated orimproperly modified). Treatment includes disorders characterized byaberrant expression or activity of the nucleic acid. In addition,disorders that are influenced by the lipase may also be treated.Examples of such disorders are disclosed here in.

Alternatively, a modulator for lipase nucleic acid expression can be asmall molecule or drug identified using the screening assays describedherein as long as the drug or small molecule inhibits the lipase nucleicacid expression.

The lipase polynucleotides are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe lipase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

Monitoring can be, for example, as follows: (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a specified mRNA orgenomic DNA of the invention in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the mRNA or genomic DNAin the post-administration samples; (v) comparing the level ofexpression or activity of the mRNA or genomic DNA in thepre-administration sample with the mRNA or genomic DNA in thepost-administration sample or samples; and (vi) increasing or decreasingthe administration of the agent to the subject accordingly.

The lipase polynucleotides are also useful in diagnostic assays forqualitative changes in lipase nucleic acid, and particularly inqualitative changes that lead to pathology. The polynucleotides can beused to detect mutations in lipase genes and gene expression productssuch as mRNA. The polynucleotides can be used as hybridization probes todetect naturally-occurring genetic mutations in the lipase gene andthereby to determine whether a subject with the mutation is at risk fora disorder caused by the mutation. Mutations include deletion, addition,or substitution of one or more nucleotides in the gene, chromosomalrearrangement, such as inversion or transposition, modification ofgenomic DNA, such as aberrant methylation patterns or changes in genecopy number, such as amplification. Detection of a mutated form of thelipase gene associated with a dysfunction provides a diagnostic tool foran active disease or susceptibility to disease when the disease resultsfrom overexpression, underexpression, or altered expression of a lipase.

Mutations in the lipase gene can be detected at the nucleic acid levelby a variety of techniques. Genomic DNA can be analyzed directly or canbe amplified by using PCR prior to analysis. RNA or cDNA can be used inthe same way.

In certain embodiments, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

It is anticipated that PCR and/or LCR may be desirable to use as apreliminary amplification step in conjunction with any of the techniquesused for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well-known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

Alternatively, mutations in a lipase gene can be directly identified,for example, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site.

Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method.

Furthermore, sequence differences between a mutant lipase gene and awild-type gene can be determined by direct DNA sequencing. A variety ofautomated sequencing procedures can be utilized when performing thediagnostic assays (1995) Biotechniques 19:448), including sequencing bymass spectrometry (see, e.g., PCT International Publication No. WO94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffinet al. (1993) Appl. Biochem. Biotechnol 38:147-159).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science 230:1242);Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992) Meth. Enzymol.217:286-295), electrophoretic mobility of mutant and wild type nucleicacid is compared (Orita et al. (1989) PNAS 86:2766; Cotton et al. (1993)Mutat. Res. 285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech.Appl. 9:73-79), and movement of mutant or wild-type fragments inpolyacrylamide gels containing a gradient of denaturant is assayed usingdenaturing gradient gel electrophoresis (Myers et al. (1985) Nature313:495). The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In one embodiment, the subject method utilizesheteroduplex analysis to separate double stranded heteroduplex moleculeson the basis of changes in electrophoretic mobility (Keen et al. (1991)Trends Genet. 7:5). Examples of other techniques for detecting pointmutations include, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

In other embodiments, genetic mutations can be identified by hybridizinga sample and control nucleic acids, e.g., DNA or RNA, to high densityarrays containing hundreds or thousands of oligonucleotide probes(Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996)Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

The lipase polynucleotides are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the polynucleotides can be used tostudy the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the lipase polynucleotides described hereincan be used to assess the mutation content of the gene in an individualin order to select an appropriate compound or dosage regimen fortreatment.

Thus polynucleotides displaying genetic variations that affect treatmentprovide a diagnostic target that can be used to tailor treatment in anindividual. Accordingly, the production of recombinant cells and animalscontaining these polymorphisms allow effective clinical design oftreatment compounds and dosage regimens.

The methods can involve obtaining a control biological sample from acontrol subject, contacting the control sample with a compound or agentcapable of detecting mRNA, or genomic DNA, such that the presence ofmRNA or genomic DNA is detected in the biological sample, and comparingthe presence of mRNA or genomic DNA in the control sample with thepresence of mRNA or genomic DNA in the test sample.

The lipase polynucleotides are also useful for chromosome identificationwhen the sequence is identified with an individual chromosome and to aparticular location on the chromosome. First, the DNA sequence ismatched to the chromosome by in situ or other chromosome-specifichybridization. Sequences can also be correlated to specific chromosomesby preparing PCR primers that can be used for PCR screening of somaticcell hybrids containing individual chromosomes from the desired species.Only hybrids containing the chromosome containing the gene homologous tothe primer will yield an amplified fragment. Sublocalization can beachieved using chromosomal fragments. Other strategies includeprescreening with labeled flow-sorted chromosomes and preselection byhybridization to chromosome-specific libraries. Further mappingstrategies include fluorescence in situ hybridization, which allowshybridization with probes shorter than those traditionally used.Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on the chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

The lipase polynucleotides can also be used to identify individualsbased on small biological samples. This can be done for example usingrestriction fragment-length polymorphism (RFLP) to identify anindividual. Thus, the polynucleotides described herein are useful as DNAmarkers for RFLP (See U.S. Pat. No. 5,272,057).

Furthermore, the lipase sequence can be used to provide an alternativetechnique, which determines the actual DNA sequence of selectedfragments in the genome of an individual. Thus, the lipase sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify DNAfrom an individual for subsequent sequencing.

Panels of corresponding DNA sequences from individuals prepared in thismanner can provide unique individual identifications, as each individualwill have a unique set of such DNA sequences. It is estimated thatallelic variation in humans occurs with a frequency of about once pereach 500 bases. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. The lipase sequences can be used to obtain such identificationsequences from individuals and from tissue. The sequences representunique fragments of the human genome. Each of the sequences describedherein can, to some degree, be used as a standard against which DNA froman individual can be compared for identification purposes.

If a panel of reagents from the sequences is used to generate a uniqueidentification database for an individual, those same reagents can laterbe used to identify tissue from that individual. Using the uniqueidentification database, positive identification of the individual,living or dead, can be made from extremely small tissue samples.

The lipase polynucleotides can also be used in forensic identificationprocedures. PCR technology can be used to amplify DNA sequences takenfrom very small biological samples, such as a single hair follicle, bodyfluids (e.g. blood, saliva, or semen). The amplified sequence can thenbe compared to a standard allowing identification of the origin of thesample.

The lipase polynucleotides can thus be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e. another DNA sequence that is unique to a particularindividual). As described above, actual base sequence information can beused for identification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to thenoncoding region are particularly useful since greater polymorphismoccurs in the noncoding regions, making it easier to differentiateindividuals using this technique.

The lipase polynucleotides can further be used to provide polynucleotidereagents, e.g., labeled or labelable probes which can be used in, forexample, an in situ hybridization technique, to identify a specifictissue. This is useful in cases in which a forensic pathologist ispresented with a tissue of unknown origin. Panels of lipase probes canbe used to identify tissue by species and/or by organ type.

In a similar fashion, these primers and probes can be used to screentissue culture for contamination (i.e. screen for the presence of amixture of different types of cells in a culture).

Alternatively, the lipase polynucleotides can be used directly to blocktranscription or translation of lipase gene sequences by means ofantisense or ribozyme constructs. Thus, in a disorder characterized byabnormally high or undesirable lipase gene expression, nucleic acids canbe directly used for treatment.

The lipase polynucleotides are thus useful as antisense constructs tocontrol lipase gene expression in cells, tissues, and organisms. A DNAantisense polynucleotide is designed to be complementary to a region ofthe gene involved in transcription, preventing transcription and henceproduction of lipase protein. An antisense RNA or DNA polynucleotidewould hybridize to the mRNA and thus block translation of mRNA intolipase protein.

Examples of antisense molecules useful to inhibit nucleic acidexpression include antisense molecules complementary to a fragment ofthe 5′ untranslated region of SEQ ID NO:2 which also includes the startcodon and antisense molecules which are complementary to a fragment ofthe 3′ untranslated region of SEQ ID NO:2.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of lipase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired lipase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the lipase protein.

The lipase polynucleotides also provide vectors for gene therapy inpatients containing cells that are aberrant in lipase gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired lipase protein totreat the individual.

The invention also encompasses kits for detecting the presence of alipase nucleic acid in a biological sample. For example, the kit cancomprise reagents such as a labeled or labelable nucleic acid or agentcapable of detecting lipase nucleic acid in a biological sample; meansfor determining the amount of lipase nucleic acid in the sample; andmeans for comparing the amount of lipase nucleic acid in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect lipase mRNA or DNA.

Computer Readable Means

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exists in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. The skilled artisan can readily adopt anyof the presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

For example, software which implements the BLAST (Altschul et al. (1990)J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem.17:203-207) search algorithms on a Sybase system can be used to identifyopen reading frames (ORFs) of the sequences of the invention whichcontain homology to ORFs or proteins from other libraries. Such ORFs areprotein encoding fragments and are useful in producing commerciallyimportant proteins such as enzymes used in various reactions and in theproduction of commercially useful metabolites.

Vectors/Host Cells

The invention also provides vectors containing the lipasepolynucleotides. The term “vector” refers to a vehicle, preferably anucleic acid molecule that can transport the lipase polynucleotides.When the vector is a nucleic acid molecule, the lipase polynucleotidesare covalently linked to the vector nucleic acid. With this aspect ofthe invention, the vector includes a plasmid, single or double strandedphage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of the lipasepolynucleotides. Alternatively, the vector may integrate into the hostcell genome and produce additional copies of the lipase polynucleotideswhen the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the lipasepolynucleotides. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the lipase polynucleotides such thattranscription of the polynucleotides is allowed in a host cell. Thepolynucleotides can be introduced into the host cell with a separatepolynucleotide capable of affecting transcription. Thus, the secondpolynucleotide may provide a trans-acting factor interacting with thecis-regulatory control region to allow transcription of the lipasepolynucleotides from the vector. Alternatively, a trans-acting factormay be supplied by the host cell. Finally, a trans-acting factor can beproduced from the vector itself.

It is understood, however, that in some embodiments, transcriptionand/or translation of the lipase polynucleotides can occur in acell-free system.

The regulatory sequence to which the polynucleotides described hereincan be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

A variety of expression vectors can be used to express a lipasepolynucleotide. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The lipase polynucleotides can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate polynucleotide can be introducedinto an appropriate host cell for propagation or expression usingwell-known techniques. Bacterial cells include, but are not limited to,E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cellsinclude, but are not limited to, yeast, insect cells such as Drosophila,animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the polypeptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the lipase polypeptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired polypeptide can ultimately beseparated from the fusion moiety. Proteolytic enzymes include, but arenot limited to, factor Xa, thrombin, and enterokinase. Typical fusionexpression vectors include pGEX (Smith et al. (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d(Studier et al. (1990) Gene Expression Technology: Methods in Enzymology185:60-89).

Recombinant protein expression can be maximized in a host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S. (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. 119-128). Alternatively, the sequenceof the polynucleotide of interest can be altered to provide preferentialcodon usage for a specific host cell, for example E. coli. (Wada et al.(1992) Nucleic Acids Res. 20:2111-2118).

The lipase polynucleotides can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.6:229-234), pMFa (Kurjan et al. (1982) Cell 30:933-943), pJRY88 (Schultzet al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The lipase polynucleotides can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow et al. (1989) Virology170:31-39).

In certain embodiments of the invention, the polynucleotides describedherein are expressed in mammalian cells using mammalian expressionvectors. Examples of mammalian expression vectors include pCDM8 (Seed,B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187-195).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the lipase polynucleotides. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of thepolynucleotides described herein. These are found for example inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning.: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the lipase polynucleotides can be introduced either alone orwith other polynucleotides that are not related to the lipasepolynucleotides such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe lipase polynucleotide vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe polynucleotides described herein or may be on a separate vector.Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the polypeptide is desired, appropriate secretionsignals are incorporated into the vector. The signal sequence can beendogenous to the lipase polypeptides or heterologous to thesepolypeptides.

Where the polypeptide is not secreted into the medium, the protein canbe isolated from the host cell by standard disruption procedures,including freeze thaw, sonication, mechanical disruption, use of lysingagents and the like. The polypeptide can then be recovered and purifiedby well-known purification methods including ammonium sulfateprecipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the polypeptides described herein, the polypeptides canhave various glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, thepolypeptides may include an initial modified methionine in some cases asa result of a host-mediated process.

Uses of Vectors and Host Cells

It is understood that “host cells” and “recombinant host cells” refernot only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

The host cells expressing the polypeptides described herein, andparticularly recombinant host cells, have a variety of uses. First, thecells are useful for producing lipase proteins or polypeptides that canbe further purified to produce desired amounts of lipase protein orfragments. Thus, host cells containing expression vectors are useful forpolypeptide production.

Host cells are also useful for conducting cell-based assays involvingthe lipase or lipase fragments. Thus, a recombinant host cell expressinga native lipase is useful to assay for compounds that stimulate orinhibit lipase function. This includes disappearance of substrate(triglycerides, phospholipids, lipoproteins), appearance of end product(fatty acids), and the various other molecular functions describedherein that include, but are not limited to, substrate recognition,substrate binding, subunit association, and interaction with othercellular components. Modulation of gene expression can occur at thelevel of transcription or translation.

Host cells are also useful for identifying lipase mutants in which thesefunctions are affected. If the mutants naturally occur and give rise toa pathology, host cells containing the mutations are useful to assaycompounds that have a desired effect on the mutant lipase (for example,stimulating or inhibiting function) which may not be indicated by theireffect on the native lipase.

Recombinant host cells are also useful for expressing the chimericpolypeptides described herein to assess compounds that activate orsuppress activation or alter specific function by means of aheterologous domain, segment, site, and the like, as disclosed herein.

Further, mutant lipase can be designed in which one or more of thevarious functions is engineered to be increased or decreased, forexample, substrate binding activity or the catalytic activity of thelipase, and used to augment or replace lipase proteins in an individual.Thus, host cells can provide a therapeutic benefit by replacing anaberrant lipase or providing an aberrant lipase that provides atherapeutic result. In one embodiment, the cells provide lipase that areabnormally active.

In another embodiment, the cells provide lipase that are abnormallyinactive. These lipases can compete with endogenous lipase polypeptidesin the individual.

In another embodiment, cells expressing lipase that cannot be activated,are introduced into an individual in order to compete with endogenouslipases for its various substrates. For example, in the case in whichexcessive lipase or analog is part of a treatment modality, it may benecessary to inactivate this molecule at a specific point in treatment.Providing cells that compete for the molecule, but which cannot beaffected by lipase activation would be beneficial.

Homologously recombinant host cells can also be produced that allow thein situ alteration of endogenous lipase polynucleotide sequences in ahost cell genome. The host cell includes, but is not limited to, astable cell line, cell in vivo, or cloned microorganism. This technologyis more fully described in WO 93/09222, WO 91/12650, WO 91/06667, U.S.Pat. Nos. 5,272,071, and 5,641,670. Briefly, specific polynucleotidesequences corresponding to the lipase polynucleotides or sequencesproximal or distal to a lipase gene are allowed to integrate into a hostcell genome by homologous recombination where expression of the gene canbe affected. In one embodiment, regulatory sequences are introduced thateither increase or decrease expression of an endogenous sequence.Accordingly, a lipase can be produced in a cell not normally producingit. Alternatively, increased expression of lipase can be effected in acell normally producing the protein at a specific level. Further,expression can be decreased or eliminated by introducing a specificregulatory sequence. The regulatory sequence can be heterologous to thelipase protein sequence or can be a homologous sequence with a desiredmutation that affects expression. Alternatively, the entire gene can bedeleted. The regulatory sequence can be specific to the host cell orcapable of functioning in more than one cell type. Still further,specific mutations can be introduced into any desired region of the geneto produce mutant lipase proteins. Such mutations could be introduced,for example, into specific functional regions such as the triglycerideor phospholipid-binding site.

In one embodiment, the host cell can be a fertilized oocyte or embryonicstem cell that can be used to produce a transgenic animal containing thealtered lipase gene. Alternatively, the host cell can be a stem cell orother early tissue precursor that gives rise to a specific subset ofcells and can be used to produce transgenic tissues in an animal. Seealso Thomas et al., Cell 51:503 (1987) for a description of homologousrecombination vectors. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedgene has homologously recombined with the endogenous lipase gene isselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos. WO 90/11354; WO 91/01140; andWO 93/04169.

The genetically engineered host cells can be used to produce non-humantransgenic animals. A transgenic animal is preferably a mammal, forexample a rodent, such as a rat or mouse, in which one or more of thecells of the animal include a transgene. A transgene is exogenous DNAwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal inone or more cell types or tissues of the transgenic animal. Theseanimals are useful for studying the function of a lipase protein andidentifying and evaluating modulators of lipase protein activity.

Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

In one embodiment, a host cell is a fertilized oocyte or an embryonicstem cell into which a lipase polynucleotide sequences have beenintroduced.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the lipase nucleotidesequences can be introduced as a transgene into the genome of anon-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the lipase protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems, which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepolypeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could affect, for example,binding, activation, and protein turnover, may not be evident from invitro cell-free or cell-based assays. Accordingly, it is useful toprovide non-human transgenic animals to assay in vivo lipase function,including substrate interaction, the effect of specific mutant on lipasefunction and substrate interaction, and the effect of chimeric lipases.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or more lipasefunctions.

In general, methods for producing transgenic animals include introducinga nucleic acid sequence according to the present invention, the nucleicacid sequence capable of expressing the lipase in a transgenic animal,into a cell in culture or in vivo. When introduced in vivo, the nucleicacid is introduced into an intact organism such that one or more celltypes and, accordingly, one or more tissue types, express the nucleicacid encoding the lipase. Alternatively, the nucleic acid can beintroduced into virtually all cells in an organism by transfecting acell in culture, such as an embryonic stem cell, as described herein forthe production of transgenic animals, and this cell can be used toproduce an entire transgenic organism. As described, in a furtherembodiment, the host cell can be a fertilized oocyte. Such cells arethen allowed to develop in a female foster animal to produce thetransgenic organism.

Pharmaceutical Compositions

The lipase nucleic acid molecules, protein modulators of the protein,and antibodies (also referred to herein as “active compounds”) can beincorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallycomprise the nucleic acid molecule, protein, modulator, or antibody anda pharmaceutically acceptable carrier.

The term “administer” is used in its broadest sense and includes anymethod of introducing the compositions of the present invention into asubject. This includes producing polypeptides or polynucleotides in vivoas by transcription or translation, in vivo, of polynucleotides thathave been exogenously introduced into a subject. Thus, polypeptides ornucleic acids produced in the subject from the exogenous compositionsare encompassed in the term “administer.”

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a lipase protein or anti-lipase antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the purview of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

This invention may be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will fully convey theinvention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 337<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1Met Asp Leu Asp Val Val Asn Met Phe Val Il #e Ala Gly Gly Thr Leu 1               5   #                10   #                15Ala Ile Pro Ile Leu Ala Phe Val Ala Ser Ph #e Leu Leu Trp Pro Ser            20       #            25       #            30Ala Leu Ile Arg Ile Tyr Tyr Trp Tyr Trp Ar #g Arg Thr Leu Gly Met        35           #        40           #        45Gln Val Arg Tyr Val His His Glu Asp Tyr Gl #n Phe Cys Tyr Ser Phe    50               #    55               #    60Arg Gly Arg Pro Gly His Lys Pro Ser Ile Le #u Met Leu His Gly Phe65                   #70                   #75                   #80Ser Ala His Lys Asp Met Trp Leu Ser Val Va #l Lys Phe Leu Pro Lys                85   #                90   #                95Asn Leu His Leu Val Cys Val Asp Met Pro Gl #y His Glu Gly Thr Thr            100       #           105       #           110Arg Ser Ser Leu Asp Asp Leu Ser Ile Asp Gl #y Gln Val Lys Arg Ile        115           #       120           #       125His Gln Phe Val Glu Cys Leu Lys Leu Asn Ly #s Lys Pro Phe His Leu    130               #   135               #   140Val Gly Thr Ser Met Gly Gly Gln Val Ala Gl #y Val Tyr Ala Ala Tyr145                 1 #50                 1 #55                 1 #60Tyr Pro Ser Asp Val Ser Ser Leu Cys Leu Va #l Cys Pro Ala Gly Leu                165   #               170   #               175Gln Tyr Ser Thr Asp Asn Gln Phe Val Gln Ar #g Leu Lys Glu Leu Gln            180       #           185       #           190Gly Ser Ala Ala Val Glu Lys Ile Pro Leu Il #e Pro Ser Thr Pro Glu        195           #       200           #       205Glu Met Ser Glu Met Leu Gln Leu Cys Ser Ty #r Val Arg Phe Lys Val    210               #   215               #   220Pro Gln Gln Ile Leu Gln Gly Leu Val Asp Va #l Arg Ile Pro His Asn225                 2 #30                 2 #35                 2 #40Asn Phe Tyr Arg Lys Leu Phe Leu Glu Ile Va #l Ser Glu Lys Ser Arg                245   #               250   #               255Tyr Ser Leu His Gln Asn Met Asp Lys Ile Ly #s Val Pro Thr Gln Ile            260       #           265       #           270Ile Trp Gly Lys Gln Asp Gln Val Leu Asp Va #l Ser Gly Ala Asp Met        275           #       280           #       285Leu Ala Lys Ser Ile Ala Asn Cys Gln Val Gl #u Leu Leu Glu Asn Cys    290               #   295               #   300Gly His Ser Val Val Met Glu Arg Pro Arg Ly #s Thr Ala Lys Leu Ile305                 3 #10                 3 #15                 3 #20Ile Asp Phe Leu Ala Ser Val His Asn Thr As #p Asn Asn Lys Lys Leu                325   #               330   #               335 Asp<210> SEQ ID NO 2 <211> LENGTH: 1964 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (164)...(1174) <400> SEQUENCE: 2cacgcgtccg gctgggctgg gcgccggagc tgggagcggc gcgggtagga gc#ccggcggc     60aggtcccagc ccggggctag agaccgaggg ccggggtccg ggcccggcgg cg#ggacccag    120 gcggttgagg ctggtcagga gtcagccagc ctgaaagagc agg atg ga#t ctt gat      175                    #                  #            Met Asp Leu As #p                    #                  #             1 gtg gtt aac atg ttt gtg att gcg ggc ggc ac#g ctg gcc atc cca atc      223Val Val Asn Met Phe Val Ile Ala Gly Gly Th #r Leu Ala Ile Pro Ile 5                  #  10                 #  15                 #  20ctg gca ttt gtg gct tca ttt ctt ctg tgg cc#t tca gca ctg ata aga      271Leu Ala Phe Val Ala Ser Phe Leu Leu Trp Pr #o Ser Ala Leu Ile Arg                 25  #                 30  #                 35atc tat tat tgg tac tgg cgg agg aca ttg gg#c atg caa gtc cgc tat      319Ile Tyr Tyr Trp Tyr Trp Arg Arg Thr Leu Gl #y Met Gln Val Arg Tyr             40      #             45      #             50gtt cac cat gaa gac tat cag ttc tgt tat tc#c ttc cgg ggc agg cct      367Val His His Glu Asp Tyr Gln Phe Cys Tyr Se #r Phe Arg Gly Arg Pro         55          #         60          #         65ggg cac aaa ccc tcc atc ctc atg ctc cac gg#a ttc tct gcc cac aag      415Gly His Lys Pro Ser Ile Leu Met Leu His Gl #y Phe Ser Ala His Lys     70              #     75              #     80gat atg tgg ctc agt gtg gtc aag ttc ctt cc#a aag aac ctg cac ttg      463Asp Met Trp Leu Ser Val Val Lys Phe Leu Pr #o Lys Asn Leu His Leu 85                  # 90                  # 95                  #100gtc tgc gtg gac atg cca gga cat gag ggc ac#c acc cgc tcc tcc ctg      511Val Cys Val Asp Met Pro Gly His Glu Gly Th #r Thr Arg Ser Ser Leu                105   #               110   #               115gat gac ctg tcc ata gat ggg caa gtt aag ag#g ata cac cag ttt gta      559Asp Asp Leu Ser Ile Asp Gly Gln Val Lys Ar #g Ile His Gln Phe Val            120       #           125       #           130gaa tgc ctg aag ctg aac aaa aaa cct ttc ca#c ctg gta ggc acc tcc      607Glu Cys Leu Lys Leu Asn Lys Lys Pro Phe Hi #s Leu Val Gly Thr Ser        135           #       140           #       145atg ggt ggc cag gtg gct ggg gtg tat gct gc#t tac tac cca tcg gat      655Met Gly Gly Gln Val Ala Gly Val Tyr Ala Al #a Tyr Tyr Pro Ser Asp    150               #   155               #   160gtc tcc agc ctg tgt ctc gtg tgt cct gct gg#c ctg cag tac tca act      703Val Ser Ser Leu Cys Leu Val Cys Pro Ala Gl #y Leu Gln Tyr Ser Thr165                 1 #70                 1 #75                 1 #80gac aat caa ttt gta caa cgg ctc aaa gaa ct#g cag ggc tct gcc gcc      751Asp Asn Gln Phe Val Gln Arg Leu Lys Glu Le #u Gln Gly Ser Ala Ala                185   #               190   #               195gtg gag aag att ccc ttg atc ccg tct acc cc#a gaa gag atg agt gaa      799Val Glu Lys Ile Pro Leu Ile Pro Ser Thr Pr #o Glu Glu Met Ser Glu            200       #           205       #           210atg ctt cag ctc tgc tcc tat gtc cgc ttc aa#g gtg ccc cag cag atc      847Met Leu Gln Leu Cys Ser Tyr Val Arg Phe Ly #s Val Pro Gln Gln Ile        215           #       220           #       225ctg caa ggc ctt gtc gat gtc cgc atc cct ca#t aac aac ttc tac cga      895Leu Gln Gly Leu Val Asp Val Arg Ile Pro Hi #s Asn Asn Phe Tyr Arg    230               #   235               #   240aag ttg ttt ttg gaa atc gtc agt gag aag tc#c aga tac tct ctc cat      943Lys Leu Phe Leu Glu Ile Val Ser Glu Lys Se #r Arg Tyr Ser Leu His245                 2 #50                 2 #55                 2 #60cag aac atg gac aag atc aag gtt ccg acg ca#g atc atc tgg ggg aaa      991Gln Asn Met Asp Lys Ile Lys Val Pro Thr Gl #n Ile Ile Trp Gly Lys                265   #               270   #               275caa gac cag gtg ctg gat gtg tct ggg gca ga#c atg ttg gcc aag tca     1039Gln Asp Gln Val Leu Asp Val Ser Gly Ala As #p Met Leu Ala Lys Ser            280       #           285       #           290att gcc aac tgc cag gtg gag ctt ctg gaa aa#c tgt ggg cac tca gta     1087Ile Ala Asn Cys Gln Val Glu Leu Leu Glu As #n Cys Gly His Ser Val        295           #       300           #       305gtg atg gaa aga ccc agg aag aca gcc aag ct#c ata atc gac ttt tta     1135Val Met Glu Arg Pro Arg Lys Thr Ala Lys Le #u Ile Ile Asp Phe Leu    310               #   315               #   320gct tct gtg cac aac aca gac aac aac aag aa#g ctg gac tgaggccccg      1184Ala Ser Val His Asn Thr Asp Asn Asn Lys Ly #s Leu Asp325                 3 #30                 3 #35actgcagcct gcattctgca cacagcatct gctcccatcc cccaagtctg ac#gcagccac   1244cactctcagg gatcctgccc caaatgcggt cggagcgcca gtgaccctga gg#aagcccgt   1304cccttatccc tggtatccac ggttccccag agctttgggg accacgcgaa aa#cctccaag   1364atatttttca caaaatagaa actcatatgg aacaaaataa gaaaccccag cc#atgaaatc   1424taccatgaag tcttcaagtt catgtcactg agaagcttgt gcaaagcagc ca#ccttggac   1484cataattaaa tcaaggacat tttctttgag acattcctta tagttggaga ct#caagatat   1544ttttgttgca tcaggtgtat tcccttgcat gggcagtggc ttttatagga gc#attagtcc   1604tcattcgctg aaccctgttg tttaggtcta atttaagttt tacatagaga cc#catgtatg   1664actgcagccc attggctgca agaccaggga ggaaagtggc aagctgtaga aa#atgtttac   1724acgcatggag gggcattgct ctagccctca gagcgtccgg agcagcaggg ta#catgggtg   1784ggaggttcat tcagcaccca ccagtcaggt atgttctgag tgaacccaca gc#agtcgcag   1844aatgagcacc tggcagggtg ggtttcctag gaataattta ttatttttaa aa#ataggcct   1904aataaagcaa taatgttcta gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa#aaaaaaaa   1964

That which is claimed:
 1. An isolated polypeptide having an amino acidsequence selected from the group consisting of: (a) the amino acidsequence set forth in SEQ ID NO:1; and (b) the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC as PatentDeposit Number PTA-1915.
 2. An isolated polypeptide having an amino acidsequence selected from the group consisting of: (a) the amino acidsequence of a fragment of the amino acid sequence set forth as aminoacids 36 to 337 of SEQ ID NO:1, wherein said fragment consists of atleast 35 contiguous amino acids of the amino acid sequence set forth asamino acids 36 to 337 of SEQ ID NO:1; and (b) the amino acid sequence ofa fragment of amino acids 36 to 337 of the amino acid sequence encodedby the cDNA insert of the plasmid deposited with ATCC as Patent DepositNumber PTA-1915, wherein said fragment consists of at least 100contiguous amino acids of amino acids 36 to 337 of the amino acidsequence encoded by the cDNA insert of the plasmid deposited with ATCCas Patent Deposit Number PTA-1915.
 3. A composition comprising thepolypeptide in claim 1 and a pharmaceutically-acceptable carrier.
 4. Theisolated polypeptide of claim 1, wherein said polypeptide has the aminoacid sequence set forth in SEQ ID NO:1.
 5. The isolated polypeptide ofclaim 2, wherein said polypeptide has the amino acid sequence of afragment of the amino acid sequence set forth as amino acids 36 to 337of SEQ ID NO:1, wherein said fragment consists of at least 35 contiguousamino acids of the amino acid sequence set forth as amino acids 36 to337 of SEQ ID NO:1.
 6. The isolated polypeptide of claim 2, wherein saidpolypeptide has the amino acid sequence of a fragment of amino acids 36to 337 of the amino acid sequence encoded by the cDNA insert of theplasmid deposited with ATCC as Patent Deposit Number PTA-1915, whereinsaid fragment consists of at least 100 contiguous amino acids of aminoacids 36 to 337 of the amino acid sequence encoded byte cDNA insert ofthe plasmid deposited with ATCC as Patent Deposit Number PTA-1915.